The herpes simplex virus type 1 thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and to image intracellular molecular events and cell trafficking in living subjects. The expression of these PRGs can be imaged using 18F- or 124I-radiolabeled acycloguanosine or pyrimidine analog PET reporter probes (PRPs). This protocol describes the procedures for imaging HSV1-tk or HSV1-sr39tk PRG expression in living subjects with the acycloguanosine analog 9-4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG). [18F]FHBG is a high-affinity substrate for the HSV1-sr39TK enzyme with relatively low affinity for mammalian TK enzymes, resulting in improved detection sensitivity. Furthermore, [18F]FHBG is approved by the US Food and Drug Administration as an investigational new imaging agent and has been shown to detect HSV1-tk transgene expression in the liver tumors of patients. MicroPET imaging of each small animal can be completed in approximately 1.5 h, and each patient imaging session takes approximately 3 h.
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Yaghoubi, S.S. & Gambhir., S.S. Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro. Nat. Protocols. 1, 2137–2142 (2006).
Saito, Y. et al. Quantitative autoradiographic mapping of herpes simplex virus encephalitis with radiolabeled antiviral drug. Science 217, 1151–1153 (1982).
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).
Gambhir, S.S. et al. Imaging of adenoviral-directed herpes simplex virus type 1 thymidine kinase reporter gene expression in mice with radiolabeled ganciclovir. J. Nucl. Med. 39, 2003–2011 (1998).
Ponomarev, V. et al. A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging. Eur. J. Nucl. Med. Mol. Imaging 31, 740–751 (2004).
Jacobs, A. et al. Positron-emission tomography of vector-mediated gene expression in gene therapy for gliomas. Lancet 358, 727–729 (2001).
Jacobs, A. et al. Positron emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo. Cancer Res. 61, 2983–2995 (2001).
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).
Iyer, M. et al. 8-[18F]fluoropenciclovir: an improved reporter probe for imaging HSV1-tk reporter gene expression in vivo using PET. J. Nucl. Med. 42, 96–105 (2001).
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).
Gambhir, S.S. et al. Imaging transgene expression with radionuclide imaging technologies. Neoplasia 2, 118–138 (2000).
Alauddin, M.M. & Conti, P.S. Synthesis and preliminary evaluation of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG): a new potential imaging agent for viral infection and gene therapy using PET. Nucl. Med. Biol. 25, 175–180 (1998).
Alauddin, M.M., Shahinian, A., Gordon, E.M., Bading, J.R. & Conti, P.S. Preclinical evaluation of the penciclovir analog 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine for in vivo measurement of suicide gene expression with PET. J. Nucl. Med. 42, 1682–1690 (2001).
Black, M.E., Kokoris, M.S. & Sabo, P. Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve prodrug-mediated tumor cell killing. Cancer Res. 61, 3022–3026 (2001).
Black, M.E., Newcomb, T.G., Wilson, H.M. & Loeb, L.A. Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy. Proc. Natl. Acad. Sci. USA 93, 3525–3529 (1996).
Min, J.-J., Iyer, M. & Gambhir, S.S. Comparison of [18F]FHBG and [14C]FIAU for imaging of HSV1-tk reporter gene expression: adenoviral infection vs stable transfection. Eur. J. Nucl. Med. Mol. Imaging 30, 1547–1560 (2003).
Tjuvajev, J.G. et al. Comparison of radiolabeled nucleoside probes (FIAU, FHBG, and FHPG) for PET imaging of HSV1-tk gene expression. J. Nucl. Med. 43, 1072–1083 (2002).
Alauddin, M.M., Shahinian, A., Gordon, E.M. & Conti, P.S. Direct comparison of radiolabeled probes FMAU, FHBG, and FHPG as PET imaging agents for HSV1-tk expression in a human breast cancer model. Mol. Imaging 3, 76–84 (2004).
Buursma, A.R. et al. 18F-FEAU as a radiotracer for herpes simplex virus thymidine kinase gene expression: in-vitro comparison with other PET tracers. Nucl. Med. Commun. 27, 25–30 (2006).
Yaghoubi, S.S. et al. Human pharmacokinetic and dosimetry studies of [18F]FHBG: a reporter probe for imaging herpes simplex virus type-1 thymidine kinase reporter gene expression. J. Nucl. Med. 42, 1225–1234 (2001).
Yaghoubi, S.S. et al. Preclinical safety evaluation of 18F-FHBG: a PET reporter probe for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk's expression. J. Nucl. Med. 47, 706–715 (2006).
Yaghoubi, S.S. et al. Imaging progress of herpes simplex virus type 1 thymidine kinase suicide gene therapy in living subjects with positron emission tomography. Cancer Gene Ther. 12, 329–339 (2005).
Penuelas, I. et al. Positron emission tomography imaging of adenoviral-mediated transgene expression in liver cancer patients. Gastroenterology 128, 1787–1795 (2005).
Ponde, D.E., Dence, C.S., Schuster, D.P. & Welch, M.J. Rapid and reproducible radiosynthesis of [18F]FHBG. Nucl. Med. Biol. 31, 133–138 (2004).
Shiue, G.G. et al. A simplified one-pot synthesis of 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG) and 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) for gene therapy. Nucl. Med. Biol. 28, 875–883 (2001).
Penuelas, I. et al. A fully automated one pot synthesis of 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine for gene therapy studies. Mol. Imaging Biol. 4, 415–424 (2003).
Hudson, H.M. & Larkin, R.S. Accelerated image reconstruction using ordered subsets of projection data. IEEE Trans. Med. Imaging 13, 601–609 (1994).
Loening, A.M. & Gambhir, S.S. AMIDE: a free software tool for multimodality medical image analysis. Mol. Imaging 2, 131–137 (2003).
Green, L.A. et al. A tracer kinetic model for 18F-FHBG for quantitating herpes simplex virus type 1 thymidine kinase reporter gene expression in living animals using PET. J. Nucl. Med. 45, 1560–1570 (2004).
Liang, Q. et al. Noninvasive, repetitive, quantitative measurement of gene expression from a bicistronic message by positron emission tomography, following gene transfer with adenovirus. Mol. Ther. 6, 73–82 (2002).
Yaghoubi, S.S. et al. Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors. Gene Ther. 8, 1072–1080 (2001).
Sundaresan, G. et al. MicroPET imaging of Cre-loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene. Gene Ther. 11, 609–618 (2004).
De, A., Lewis, X.Z. & Gambhir, S.S. Noninvasive imaging of lentiviral-mediated reporter gene expression in living mice. Mol. Ther. 7, 681–691 (2003).
Ray, P., De, A., Min, J.J., Tsien, R.Y. & Gambhir, S.S. Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res. 64, 1323–1330 (2004).
Pantuck, A.J. et al. Optimizing prostate cancer suicide gene therapy using herpes simplex virus thymidine kinase active site variants. Hum. Gene Ther. 13, 777–789 (2002).
Pantuck, A.J. et al. CL1-SR39: a noninvasive molecular imaging model of prostate cancer suicide gene therapy using positron emission tomography. J. Urol. 168, 1193–1198 (2002).
Sun, X. et al. Quantitative imaging of gene induction in living animals. Gene Ther. 8, 1572–1579 (2001).
Yu, Y. et al. Quantification of target gene expression by imaging reporter gene expression in living animals. Nat. Med. 6, 933–937 (2000).
Yang, H., Berger, F., Tran, C., Gambhir, S.S. & Sawyers, C.L. MicroPET imaging of prostate cancer in LNCAP-SR39TK-GFP mouse xenografts. Prostate 55, 39–47 (2003).
Wang, Y. et al. Noninvasive monitoring of target gene expression by imaging reporter gene expression in living animals using improved bicistronic vectors. J. Nucl. Med. 46, 667–674 (2005).
Richard, J.-C., Factor, P., Welch, L.C. & Schuster, D.P. Imaging the spatial distribution of transgene expression in the lungs with positron emission tomography. Gene Ther. 10, 2074–2080 (2003).
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).
Sen, L. et al. Noninvasive imaging of ex vivo intracoronarily delivered nonviral therapeutic transgene expression in heart. Mol. Ther. 12, 49–57 (2005).
Kim, Y.J., Dubey, P., Ray, P., Gambhir, S.S. & Witte, O.N. Multimodality imaging of lymphocytic migration using lentiviral-based transduction of a tri-fusion reporter gene. Mol. Imaging Biol. 6, 331–340 (2004).
Dubey, P. et al. Quantitative imaging of T cell antitumor response by positron-emission tomography. Proc. Natl. Acad. Sci. USA 100, 1232–1237 (2003).
Cao, F. et al. In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery. Circulation 113, 1005–1014 (2006).
Penuelas, I., Haberkorn, U., Yaghoubi, S. & Gambhir, S.S. Gene therapy imaging in patients for oncological applications. Eur. J. Nucl. Med. Mol. Imaging 32, S384–S403 (2005).
The authors acknowledge the following funding sources: NIH grants NCI ICMIC P50 CA114747, SAIRP R24 CA92865, P50 CA86306, R01 CA82214-01; DOE contract DE-FC03-87ER60615; RSNA Postdoctoral Fellowship in Basic Radiological Sciences.
The authors declare no competing financial interests.
Shown is a mouse microPET dynamic image set taken over the course of 60 min after injection of the tracer [18F]FHBG via tail-vein. The mouse was injected via tail-vein with 1 × 109 plaque forming units of a replication deficient adenovirus carrying the HSV1-sr39tk reporter gene 48 h prior to the small animal PET imaging study. The tracer quickly distributes out of the blood and clears via the renal system into the bladder. Tracer is a so slowly extracted from the blood and is trapped in hepatocytes that express the HSV1-sr39tk reporter gene. Some tracer also clear via the hepatobiliary system into the gastro-intestinal tract. (MPG 1098 kb)
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Yaghoubi, S., Gambhir, 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, 3069–3074 (2006). https://doi.org/10.1038/nprot.2006.459
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