A Genetically Engineered Reporter System Designed for ²H-MRI Allows Quantitative <i>In Vivo</i> Mapping of Transgene Expression

The ability to obtain quantitative spatial information on subcellular processes of deep tissues <i>in vivo</i> has been a long-standing challenge for molecular magnetic resonance imaging (MRI) approaches. This challenge remains even more so for quantifying readouts of genetically engineered MRI reporters. Here, we set to overcome this challenge with a molecular system designed to obtain quantitative ²H-MRI maps of a gene reporter. To this end, we synthesized deuterated thymidine, <i>d</i>₃-thy, with three magnetically equivalent deuterons at its methyl group (-CD₃), showing a singlet peak with a characteristic ²H-NMR frequency (δ = 1.7 ppm). The upfield 3.0 ppm offset from the chemical shift of the HDO signal (δ = 4.7 ppm) allows for spectrally resolving the two ²H NMR signals and quantifying the concentration of <i>d</i>₃-thy based on the known concentration of a tissue's HDO. Following systemic administration of <i>d</i>₃-thy, its accumulation as <i>d</i>₃-thy monophosphate in cells expressing the human thymidine kinase 1 (hTK1) transgene was mapped with ²H-MRI. The data obtained <i>in vivo</i> show the ability to use the <i>d</i>₃-thy/hTK1 pair as a reporter probe/reporter gene system to quantitatively map transgene expression with MRI. Relying on a structurally unmodified reporter probe (<i>d</i>₃-thy) to image the expression of unmutated human protein (hTK1) shows the potential of molecular imaging with ²H-MRI to monitor gene reporters and other relevant biological targets.