Noninvasive Imaging of Transgene Expression in Neurons Using Chemical Exchange Saturation Transfer MRI

Advances in gene therapy, especially for brain diseases, have created new imaging demands for noninvasive monitoring of gene expression. While reporter gene imaging using co-expression of fluorescent protein-encoding gene has been widely developed, these conventional methods face significant limitations in longitudinal in vivo applications. Magnetic resonance imaging (MRI), specifically chemical exchange saturation transfer (CEST) MRI, provides a robust noninvasive alternative that offers unlimited depth penetration, reliable spatial resolution, and specificity toward particular molecules. In this study, we explore the potential of CEST-MRI for monitoring gene expression in neurons. We designed a CEST polypeptide reporter expressing 150 arginine residues and evaluated its expression in the living brain after viral vector delivery. A longitudinal study performed at one and 2 months postinjection showed that specific CEST signal was observable. In particular, the CEST contrast exhibited distinct peaks at 0.75 and 1.75 ppm, consistent with the expected hydroxyl and guanidyl protons resonance frequencies. Histological study confirmed the specific neuronal expression of the transgene evidenced by the fluorescence signal from the td-Tomato fluorophore fused to the polypeptide. The ability to image noninvasively a neuron-specific CEST-MRI reporter gene could offer valuable insights for further developments of gene therapy for neurological disorders.