Non-invasive tracking of rAAV-mediated gene expression in the liver through a genetically encoded MRI reporter gene

Non-invasive imaging of gene expression remains a critical challenge in gene therapy. Here, we report the first in vivo demonstration of a genetically encoded chemical exchange saturation transfer (CEST) MRI reporter expressed from a recombinant adeno-associated virus (rAAV). The genetically encoded peptide-based reporter superCESTide enables detection of transgene expression without the need for exogenous contrast agents. Mice were systemically administered rAAV encoding superCESTide and fluorescence reporter tdTomato, and they were evaluated using CEST MRI, fluorescence imaging, and reverse transcription-PCR. CEST MRI revealed dose-dependent signal enhancement in the liver, with a strong correlation between CEST contrast and superCESTide expression (r = 0.75, p < 0.05). Mice receiving the highest dose (1.4 × 10¹³ vector genomes/kg) exhibited the strongest MTR_asym signal (mean = 0.0225), and histogram analyses showed distinct expression patterns. These results establish CEST MRI as a powerful, non-invasive method for monitoring rAAV-mediated transgene expression in vivo, opening new possibilities for imaging-guided gene therapy.