A Genetically Engineered Reporter System Designed for ²H-MRI Allows Quantitative In Vivo Mapping of Transgene...

The ability to obtain quantitative spatial information on subcellular processes of deep tissues in vivo 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, d₃-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 d₃-thy based on the known concentration of a tissue's HDO. Following systemic administration of d₃-thy, its accumulation as d₃-thy monophosphate in cells expressing the human thymidine kinase 1 (hTK1) transgene was mapped with ²H-MRI. The data obtained in vivo show the ability to use the d₃-thy/hTK1 pair as a reporter probe/reporter gene system to quantitatively map transgene expression with MRI. Relying on a structurally unmodified reporter probe (d₃-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.