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Synthesis of a probe for monitoring HSV1-tk reporter gene expression using chemical exchange saturation transfer MRI

Nature Protocols volume 8, pages 2380–2391 (2013)Cite this article

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Abstract

In experiments involving transgenic animals or animals treated with transgenic cells, it is important to have a method to monitor the expression of the relevant genes longitudinally and noninvasively. An MRI-based reporter gene enables monitoring of gene expression in the deep tissues of living subjects. This information can be co-registered with detailed high-resolution anatomical and functional information. We describe here the synthesis of the reporter probe, 5-methyl-5,6-dihydrothymidine (5-MDHT), which can be used for imaging of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression in rodents by MRI. The protocol also includes data acquisition and data processing routines customized for chemical exchange saturation transfer (CEST) contrast mechanisms. The dihydropyrimidine 5-MDHT is synthesized through a catalytic hydrogenation of the 5,6-double bond of thymidine to yield 5,6-dihydrothymidine, which is methylated on the C-5 position of the resulting saturated pyrimidine ring. The synthesis of 5-MDHT can be completed within 5 d, and the compound is stable for more than 1 year.

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Figure 1: Synthetic scheme for 5-MDHT.
Figure 2: CEST-MRI data acquisition and processing.
Figure 3: Histological validation of HSV1-tk expression.
Figure 4: In vivo detection of the imino proton in 9LHSV1−tk.

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References

  1. Yigit, M.V., Moore, A. & Medarova, Z. Magnetic nanoparticles for cancer diagnosis and therapy. Pharmacol. Res. 29, 1180–1188 (2012).

    Article  CAS  Google Scholar 

  2. Culver, K.W. et al. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 256, 1550–1552 (1992).

    Article  CAS  Google Scholar 

  3. Tjuvajev, J.G. et al. Imaging herpes virus thymidine kinase gene transfer and expression by positron emission tomography. Cancer Res. 58, 4333–4341 (1998).

    CAS  PubMed  Google Scholar 

  4. Tjuvajev, J.G. et al. Noninvasive imaging of herpes virus thymidine kinase gene transfer and expression: a potential method for monitoring clinical gene therapy. Cancer Res. 56, 4087–4095 (1996).

    CAS  PubMed  Google Scholar 

  5. Gambhir, S.S. et al. Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography. Proc. Natl. Acad. Sci. USA 96, 2333–2338 (1999).

    Article  CAS  Google Scholar 

  6. Gambhir, S.S. et al. A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography. Proc. Natl. Acad. Sci. USA 97, 2785–2790 (2000).

    Article  CAS  Google Scholar 

  7. Jacobs, A. et al. Positron-emission tomography of vector-mediated gene expression in gene therapy for gliomas. Lancet 358, 727–729 (2001).

    Article  CAS  Google Scholar 

  8. Yaghoubi, S.S. et al. Noninvasive detection of therapeutic cytolytic T cells with 18F-FHBG PET in a patient with glioma. Nat. Rev. Clin. Oncol. 6, 53–58 (2009).

    Article  CAS  Google Scholar 

  9. Freytag, S.O. et al. Phase I study of replication-competent adenovirus-mediated double-suicide gene therapy for the treatment of locally recurrent prostate cancer. Cancer Res. 62, 4968–4976 (2002).

    CAS  PubMed  Google Scholar 

  10. Freytag, S.O. et al. Five-year follow-up of trial of replication-competent adenovirus-mediated suicide gene therapy for treatment of prostate cancer. Mol. Ther. 15, 636–642 (2007).

    Article  CAS  Google Scholar 

  11. Miyagawa, T. et al. Imaging of HSV-tk reporter gene expression: comparison between [18F]FEAU, [18F]FFEAU, and other imaging probes. J. Nucl. Med. 49, 637–648 (2008).

    Article  CAS  Google Scholar 

  12. Sun, N., Lee, A. & Wu, J.C. Long term non-invasive imaging of embryonic stem cells using reporter genes. Nat. Protoc. 4, 1192–1201 (2009).

    Article  CAS  Google Scholar 

  13. Yaghoubi, S.S., Berger, F. & Gambhir, S.S. Studying the biodistribution of positron emission tomography reporter probes in mice. Nat. Protoc. 2, 1752–1755 (2007).

    Article  CAS  Google Scholar 

  14. Yaghoubi, S.S. & Gambhir, S.S. Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro. Nat. Protoc. 1, 2137–2142 (2006).

    Article  CAS  Google Scholar 

  15. Soghomonyan, S. et al. Molecular PET imaging of HSV1-tk reporter gene expression using [18F]FEAU. Nat. Protoc. 2, 416–423 (2007).

    Article  CAS  Google Scholar 

  16. Yaghoubi, S.S. & Gambhir, S.S. PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [18F]FHBG. Nat. Protoc. 1, 3069–3075 (2006).

    Article  CAS  Google Scholar 

  17. Burton, J.B. et al. Adenovirus-mediated gene expression imaging to directly detect sentinel lymph node metastasis of prostate cancer. Nat. Med. 14, 882–888 (2008).

    Article  CAS  Google Scholar 

  18. Hung, S.C. et al. Mesenchymal stem cell targeting of microscopic tumors and tumor stroma development monitored by noninvasive in vivo positron emission tomography imaging. Clin. Cancer Res. 11, 7749–7756 (2005).

    Article  CAS  Google Scholar 

  19. Willmann, J.K. et al. Imaging gene expression in human mesenchymal stem cells: from small to large animals. Radiology 252, 117–127 (2009).

    Article  Google Scholar 

  20. Wu, J.C., Inubushi, M., Sundaresan, G., Schelbert, H.R. & Gambhir, S.S. Positron emission tomography imaging of cardiac reporter gene expression in living rats. Circulation 106, 180–183 (2002).

    Article  Google Scholar 

  21. Lubag, A.J., De Leon-Rodriguez, L.M., Burgess, S.C. & Sherry, A.D. Noninvasive MRI of beta cell function using a Zn2+-responsive contrast agent. Proc. Natl. Acad. Sci. USA 108, 18400–18405 (2011).

    Article  CAS  Google Scholar 

  22. Loving, G.S., Mukherjee, S. & Caravan, P. Redox-activated manganese-based MR contrast agent. J. Am. Chem. Soc. 135, 4620–4623 (2013).

    Article  CAS  Google Scholar 

  23. Osborne, E.A., Jarrett, B.R., Tu, C. & Louie, A.Y. Modulation of T2 relaxation time by light-induced, reversible aggregation of magnetic nanoparticles. J. Am. Chem. Soc. 132, 5934–5935 (2010).

    Article  CAS  Google Scholar 

  24. Catanzaro, V. et al. A R2p /R1p ratiometric procedure to assess matrix metalloproteinase-2 activity by magnetic resonance imaging. Angew. Chem. Int. Ed. Engl. 52, 3926–3930 (2013).

    Article  CAS  Google Scholar 

  25. Cohen, B. et al. MRI detection of transcriptional regulation of gene expression in transgenic mice. Nat. Med. 13, 498–503 (2007).

    Article  CAS  Google Scholar 

  26. Genove, G., DeMarco, U., Xu, H., Goins, W.F. & Ahrens, E.T. A new transgene reporter for in vivo magnetic resonance imaging. Nat. Med. 11, 450–454 (2005).

    Article  CAS  Google Scholar 

  27. Gilad, A.A. et al. Artificial reporter gene providing MRI contrast based on proton exchange. Nat. Biotech. 25, 217–219 (2007).

    Article  CAS  Google Scholar 

  28. Ward, K.M., Aletras, A.H. & Balaban, R.S. A new class of contrast agents for MRI based on proton chemical exchange dependent saturation transfer (CEST). J. Magn. Reson. 143, 79–87 (2000).

    Article  CAS  Google Scholar 

  29. Sherry, A.D. & Woods, M. Chemical exchange saturation transfer contrast agents for magnetic resonance imaging. Annu. Rev. Biomed. Eng. 10, 391–411 (2008).

    Article  CAS  Google Scholar 

  30. van Zijl, P.C. & Yadav, N.N. Chemical exchange saturation transfer (CEST): what is in a name and what isn't? Magn. Reson. Med. 65, 927–948 (2011).

    Article  CAS  Google Scholar 

  31. Yoo, B. & Pagel, M.D. A PARACEST MRI contrast agent to detect enzyme activity. J. Am. Chem. Soc. 128, 14032–14033 (2006).

    Article  CAS  Google Scholar 

  32. Yoo, B., Raam, M.S., Rosenblum, R.M. & Pagel, M.D. Enzyme-responsive PARACEST MRI contrast agents: a new biomedical imaging approach for studies of the proteasome. Contrast Media Mol. Imaging 2, 189–198 (2007).

    Article  CAS  Google Scholar 

  33. Liu, G. et al. Monitoring enzyme activity using a diamagnetic chemical exchange saturation transfer magnetic resonance imaging contrast agent. J. Am. Chem. Soc. 133, 16326–16329 (2011).

    Article  CAS  Google Scholar 

  34. Liu, G. & Gilad, A.A. MRI of CEST-based reporter gene. Methods Mol. Biol. 771, 733–746 (2011).

    Article  CAS  Google Scholar 

  35. Bar-Shir, A. et al. Transforming thymidine into a magnetic resonance imaging probe for monitoring gene expression. J. Am. Chem. Soc. 135, 1617–1624 (2013).

    Article  CAS  Google Scholar 

  36. Najjar, A.M. et al. Molecular-genetic PET imaging using an HSV1-tk mutant reporter gene with enhanced specificity to acycloguanosine nucleoside analogs. J. Nucl. Med. 50, 409–416 (2009).

    Article  CAS  Google Scholar 

  37. Greenberg, M.M. Investigating nucleic acid damage processes via independent generation of reactive intermediates. Chem. Res. Toxicol. 11, 1235–1248 (1998).

    Article  CAS  Google Scholar 

  38. Teoule, R., Fouque, B. & Cadet, J. Synthesis and spectroscopic properties of two classes of 5,6-dihydrothymidine derivatives. Action on the Ehrlich's ascites cells thymidine kinase. Nucleic Acids Res. 2, 487–499 (1975).

    Article  CAS  Google Scholar 

  39. Judenhofer, M.S. et al. Simultaneous PET-MRI: a new approach for functional and morphological imaging. Nat. Med. 14, 459–465 (2008).

    Article  CAS  Google Scholar 

  40. Greenberg, M.M. et al. DNA damage induced via 5,6-dihydrothymid-5-yl in single-stranded oligonucleotides. J. Am. Chem. Soc. 119, 1828–1839 (1997).

    Article  CAS  Google Scholar 

  41. Barvian, M.R. & Greenberg, M.M. Independent generation of 5,6-dihydrothymid-5-yl and investigation of its ability to effect nucleic-acid strand scission via hydrogen-atom abstraction. J. Org. Chem. 60, 1916–1917 (1995).

    Article  CAS  Google Scholar 

  42. Greenberg, M.M. & Matray, T.J. Inhibition of Klenow fragment (exo−) catalyzed DNA polymerization by (5R)-5,6-dihydro-5-hydroxythymidine and structural analogue 5,6-dihydro-5-methylthymidine. Biochemistry 36, 14071–14079 (1997).

    Article  CAS  Google Scholar 

  43. Kim, M., Gillen, J., Landman, B.A., Zhou, J. & van Zijl, P.C. Water saturation shift referencing (WASSR) for chemical exchange saturation transfer (CEST) experiments. Magn. Reson. Med. 61, 1441–1450 (2009).

    Article  Google Scholar 

  44. Lipton, M.F., Sorensen, C.M., Sadler, A.C. & Shapiro, R.H. Convenient method for the accurate estimation of concentrations of alkyllithium reagents. J. Organomet. Chem. 186, 155–158 (1980).

    Article  CAS  Google Scholar 

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Acknowledgements

This study was supported by grant nos. U54CA151838 (US National Institutes of Health (NIH)), MSCRFF-0103-00 (Maryland Stem Cell Research Fund), NIH 2R01 NS045062 and GM-054996 (NIH). We thank M. McAllister for her assistance in editing the manuscript.

Author information

Authors and Affiliations

  1. Division of Magnetic Resonance Research, Russell H. Morgan Department of Radiology and Radiological Science, the Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Amnon Bar-Shir, Guanshu Liu, Jeff W M Bulte & Assaf A Gilad

  2. Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, the Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Amnon Bar-Shir, Jeff W M Bulte & Assaf A Gilad

  3. F.M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Institute, Baltimore, Maryland, USA

    Guanshu Liu, Jeff W M Bulte & Assaf A Gilad

  4. Department of Chemistry, the Johns Hopkins University, Baltimore, Maryland, USA

    Marc M Greenberg

  5. Department of Biomedical Engineering, the Johns Hopkins University, Baltimore, Maryland, USA

    Jeff W M Bulte

  6. Department of Chemical and Biomolecular Engineering, the Johns Hopkins University, Baltimore, Maryland, USA

    Jeff W M Bulte

  7. Department of Oncology, the Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Jeff W M Bulte

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  1. Amnon Bar-Shir
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  5. Assaf A Gilad
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Contributions

A.B.-S., J.W.M.B. and A.A.G. were responsible for the study concept, design of experiments, data analysis and results interpretation. A.B.-S. and M.M.G. performed the chemical synthesis of the CEST probe. A.B.-S. and A.A.G. performed cloning, cell transfection, cell transplantation, in vivo MRI experiments and immunofluorescence. A.B.-S., G.L. and A.A.G. processed the CEST MRI data. A.B.-S., J.W.M.B. and A.A.G. wrote the protocol.

Corresponding author

Correspondence to Assaf A Gilad.

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Bar-Shir, A., Liu, G., Greenberg, M. et al. Synthesis of a probe for monitoring HSV1-tk reporter gene expression using chemical exchange saturation transfer MRI. Nat Protoc 8, 2380–2391 (2013). https://doi.org/10.1038/nprot.2013.140

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