School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang 110016, China.
State Key Laboratory of Drug Regulatory Science, National Institutes for Food and Drug Control, No. 31 Huatuo St., Daxing District, Beijing 100050, China.
Molecules. 2024 Nov 11;29(22):5312. doi: 10.3390/molecules29225312.
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.
治疗基因的表达对于基因治疗产品的疗效至关重要。然而，现有的方法，如蛋白质水平的免疫分析或 RNA 水平的逆转录 PCR，无法准确地定量目标基因的表达活性。在此，开发了一种基于体外 RNA 编辑的报告基因检测方法来检测特定的 mRNA。设计的传感器 RNA 可以特异性识别靶 mRNA，并且由于内源性腺苷脱氨酶作用于 RNA 介导的 RNA 编辑，报告基因以剂量依赖性方式被激活。值得注意的是，针对不同区域（包括感兴趣的基因、标签序列和 3'非翻译区）的所有传感器都显示出剂量依赖性反应模式。该传感器报告基因检测方法用于定量基于重组腺相关病毒的基因治疗产品的转录活性，在检测特异性、精密度（批内相对标准偏差 <15%）、准确度（90-115%回收）和线性度（R > 0.99）方面表现出优异的性能。该报告基因检测方法还可用于其他基因治疗载体，包括 mRNA 和重组慢病毒。因此，开发了一种用于评估治疗基因转录活性的强大而可靠的平台，从而为基因治疗产品的质量控制提供了有力的工具。
