Non-invasive Imaging in Gene Therapy
Authors: Jani Kristian Räty, Timo Liimatainen, Minna Unelma Kaikkonen, Olli Gröhn, Kari Jumani Airenne, Seppo Ylä-Herttuala
Journal: Molecular Therapy (2007)

Several methods are available for non-invasive imaging of gene delivery and transgene expression, including magnetic resonance imaging (MRI), single photon emission tomography (SPECT)/positron emission tomography (PET), and fluorescence and bioluminescence imaging. However, these imaging modalities differ greatly in terms of their sensitivity, cost, and ability to measure the signal. Whereas MRI can produce a resolution of approximately 50 μm, optical imaging achieves only 3–5 mm but outperforms MRI in terms of the cost of the imaging device. Similarly, SPECT and PET give a resolution of only 1–2 mm but provide for relatively easy quantitation of the signal and need only nanograms of probe, compared with the microgram or milligram levels required for MRI and optical imaging. To develop safer and more efficient gene delivery vectors, it is essential to perform rigorous in vivo experiments, to image particle biodistribution and transduction patterns, and to quantify the transgene expression profile. Differences between modalities have a significant effect on the resultant imaging resolution for gene therapy. This review describes the methodologies in use and highlights recent key approaches using the latest imaging modalities in gene therapy. Future trends in gene therapy imaging are also discussed. Several methods are available for non-invasive imaging of gene delivery and transgene expression, including magnetic resonance imaging (MRI), single photon emission tomography (SPECT)/positron emission tomography (PET), and fluorescence and bioluminescence imaging. However, these imaging modalities differ greatly in terms of their sensitivity, cost, and ability to measure the signal. Whereas MRI can produce a resolution of approximately 50 μm, optical imaging achieves only 3–5 mm but outperforms MRI in terms of the cost of the imaging device. Similarly, SPECT and PET give a resolution of only 1–2 mm but provide for relatively easy quantitation of the signal and need only nanograms of probe, compared with the microgram or milligram levels required for MRI and optical imaging. To develop safer and more efficient gene delivery vectors, it is essential to perform rigorous in vivo experiments, to image particle biodistribution and transduction patterns, and to quantify the transgene expression profile. Differences between modalities have a significant effect on the resultant imaging resolution for gene therapy. This review describes the methodologies in use and highlights recent key approaches using the latest imaging modalities in gene therapy. Future trends in gene therapy imaging are also discussed. Non-invasive in vivo imaging has emerged as an essential tool for identifying the fate of vectors in tissues, quantifying injected dose in various organs, evaluating toxicity, and analyzing therapeutic gene expression level and treatment outcome. It would be valuable to address all these points during the development of new, safe gene delivery vectors. For example, non-invasive imaging of central nervous system, tumor, and cardiovascular tissues could verify gene expression in actual target tissues, resulting in better therapeutic approaches.1Wu JC Yla-Herttuala S Human gene therapy and imaging: cardiology.Eur J Nucl Med Mol Imaging. 2005; 32 (suppl. 2): S346-S357Crossref PubMed Scopus (13) Google Scholar Current imaging methods in gene therapy can be divided into two classes: transduction and biodistribution imaging. Transduction imaging visualizes transgene-mediated protein production; biodistribution imaging visualizes the actual systemic distribution of gene delivery vectors.2Kootstra NA Verma IM Gene therapy with viral vectors.Annu Rev Pharmacol Toxicol. 2003; 43: 413-439Crossref PubMed Scopus (217) Google Scholar Studying only the transduction pattern may yield an inaccurate picture of viral biodistribution, as a virus may be able to enter into many cells but unable to express its transgenes in all cells. Depending on the treatment, this could result in unexpected side effects, including immuno-responses. Therefore, it is important to evaluate both particle kinetics and transgene expression in vivo. Imaging technologies that allow non-invasive visualization of the body are based on different forms of energy interacting with tissues. Some methods, such as computed tomography and magnetic resonance imaging (MRI), rely on energy–tissue interactions, whereas single photon emission tomography (SPECT) and positron emission tomography (PET) require the administration of reporter probes. However, in both computed tomography and MRI, exogenous contrast agents can be exploited to enhance contrast or follow biological markers. With genomics research leading to the emergence and development of molecular medicine, therapeutic agents and specialized imaging techniques are becoming increasingly important.3Bogdanov Jr A In vivo imaging in the development of gene therapy vectors.Curr Opin Mol Ther. 2003; 5: 594-602PubMed Google Scholar In general, in vivo molecular imaging is thought to reduce costs and time (e.g., via the decreased use of laboratory animals and time-consuming invasive methods).4Massoud TF Gambhir SS Molecular imaging in living subjects: seeing fundamental biological processes in a new light.Genes Dev. 2003; 17: 545-580Crossref PubMed Scopus (1848) Google Scholar The differences between the main imaging modalities are illustrated in Figure 1. MRI offers the advantages of high spatial resolution and the ability to measure more than one physiological parameter using different pulse sequences. Unlike in most other imaging modalities, no radioactivity is involved, making follow-up studies more feasible. The underlying principle of MRI is nuclear magnetic resonance: unpaired nuclear spins (usually hydrogen atoms from water or organic compounds) align themselves when they are placed in a magnetic field, forming a net effect called net magnetization. The net magnetization can be disturbed from its equilibrium state using a radio-frequency pulse. The system then starts to relax back toward an equilibrium state, with the relaxation time corresponding to the physicochemical environment. The commonly used proton MRI utilizes the nuclear magnetic resonance signal from the water proton spin during relaxation, which is sometimes augmented by contrast agents, usually transition metals with a high magnetic moment. The magnetic properties of tissues affect MR images, and differences in magnetic susceptibility can be used to provide the contrast required to form an image. Volumetric MRI is based on the contrast between a tissue of interest and normal tissue. In gene therapy applications, volumetric MRI can be used to measure the volume of a tumor. This approach provides high-resolution images with versatile contrasts and clear delineation of tumor borders. However, volumetric changes in large tumor masses take place rather late in therapy and thus cannot be used efficiently as early markers. Different types of MRI contrast can be achieved by sensitizing imaging for diffusion or measuring the apparent diffusion coefficient. The signal loss in diffusion-weighted imaging is due to Brownian motion of molecules, usually water molecules.5Kauppinen RA Monitoring cytotoxic tumour treatment response by diffusion magnetic resonance imaging and proton spectroscopy.NMR Biomed. 2002; 15: 6-17Crossref PubMed Scopus (103) Google Scholar The apparent diffusion coefficient of water has been found to increase in the early phase of anticancer therapies in several studies;5Kauppinen RA Monitoring cytotoxic tumour treatment response by diffusion magnetic resonance imaging and proton spectroscopy.NMR Biomed. 2002; 15: 6-17Crossref PubMed Scopus (103) Google Scholar,6Kettunen MI Grohn OH Tumour gene therapy monitoring using magnetic resonance imaging and spectroscopy.Curr Gene Ther. 2005; 5: 685-696Crossref PubMed Scopus (11) Google Scholar,7Ross BD Moffat BA Lawrence TS Mukherji SK Gebarski SS Quint DJ et al.Evaluation of cancer therapy using diffusion magnetic resonance imaging.Mol Cancer Ther. 2003; 2: 581-587PubMed Google Scholar an example is shown in Figure 2. For this and several other MRI parameters, the most reliable results are yielded when treatment response can be monitored from the beginning of treatment. There are several sources of static magnetic field variation in the body, such as internal magnetic susceptibility differences between tissues, foreign objects, surgical clips, and implants. All these variations can cause distortions and loss of the MR signal. The sensitivity of MRI to differences in magnetic susceptibility can also be exploited, either by enhancing endogenous MRI contrasts or by using exogenous contrast agents that can cause differences in local magnetic susceptibility. The inherent insensitivity of MRI techniques prevents the direct detection of gene delivery vectors. This can be circumvented using efficient cellular amplification strategies and exogenous MRI contrast agents.3Bogdanov Jr A In vivo imaging in the development of gene therapy vectors.Curr Opin Mol Ther. 2003; 5: 594-602PubMed Google Scholar,8Schellingerhout D Bogdanov AA Viral imaging in gene therapy noninvasive demonstration of gene delivery and expression.Neuroimaging Clin N Am. 2002; 12: 571-581Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar Contrast agents make it possible to detect gene delivery vectors and provide anatomical and morphological information, as well as information about the quantity of viral vectors or cells. However, the metabolic status of transduced cells cannot be determined with the commonly used contrast agents. Contrast agents are chemical substances introduced into the anatomical or functional regions to be imaged to increase the contrast between intact and abnormal tissues by altering relaxation times. Contrast agents used in MRI can be divided into two categories: mainly T1-affecting agents with a positive contrast (increasing the signal in T1-weighted MRI) and mainly T2-affecting agents with a negative contrast (decreasing the signal in T2(*)-weighted MRI). However, agents in both categories have some effect on both T1 and T2 relaxation times. The T1 time constant relates to longitudinal relaxation in the direction of the ambient magnetic field. It is caused by spin interactions with the surrounding lattice and is thus known as "spin-lattice" relaxation. The T2 time constant describes signal attenuation perpendicular to the ambient magnetic field, and the relaxation derives from the interaction of the spins. It is known as "spin–spin" or transverse relaxation. T1 contrast agents. Typical T1 contrast agents are low-molecular-weight compounds containing a lanthanide, such as gadolinium. As free gadolinium is toxic even in low concentrations, it is bound to a chelate (e.g., gadolinium-diethylene triamine pentaacetic acid). The first-generation MR contrast agents were based on this design and have been used to image ruptures in the blood–brain barrier. Since the relaxivity values are in the range of 5–80 (mmol/l/ms)–1, depending on, for instance, size, chemical structure, and binding sites for water, the required concentration needs to be in the order of millimoles per liter.4Massoud TF Gambhir SS Molecular imaging in living subjects: seeing fundamental biological processes in a new light.Genes Dev. 2003; 17: 545-580Crossref PubMed Scopus (1848) Google Scholar Gadolinium-tetraazacyclododecane tetraacetic acid tripositively charged spermidine residue has been used to label negatively charged plasmid DNA to visualize DNA entrapment in rabbit muscle during electroporation.9Crich SG Lanzardo S Barge A Esposito G Tei L Forni G et al.Visualization through magnetic resonance imaging of DNA internalized following "in vivo" electroporation.Mol Imaging. 2005; 4: 7-17PubMed Google Scholar Another method is to load protein cage structures, such as viral scaffolds, with a lanthanide. This method has been used with cowpea chlorotic mottle virus particles introduced as high-relaxivity MRI contrast agents to image viral particles.10Allen M Bulte JW Liepold L Basu G Zywicke HA Frank JA et al.Paramagnetic viral nanoparticles as potential high-relaxivity magnetic resonance contrast agents.Magn Reson Med. 2005; 54: 807-812Crossref PubMed Scopus (176) Google Scholar In another approach used to increase relaxivity, the expression of the LacZ marker gene was detected by exploiting the ability of enzymes to cleave carbohydrate residues from modified gadolinium chelates.11Louie AY Huber MM Ahrens ET Rothbacher U Moats R Jacobs RE et al.In vivo visualization of gene expression using magnetic resonance imaging.Nat Biotechnol. 2000; 18: 321-325Crossref PubMed Scopus (983) Google Scholar T2 contrast agents. As T2 contrast agents, different iron oxide nanoparticles have proven efficiency in biomedical imaging. Such particles usually consist of a crystalline iron oxide core, typically Fe3O4, surrounded by a dextran polymer, polyethylene glycol, or ionic substances such as citrate. Because these agents consist of solid iron, the relaxivity values can be up to 200 mmol/l/s. Clinically approved agents, such as ferumoxtran-10, provide an established method for use in gene and cell therapy to track the biodistribution of particles or cells in vivo. In general terms, ultra-small paramagnetic iron oxide (USPIO) particles have a diameter of less than 50 nm, and super-paramagnetic iron oxide (SPIO) particles have a diameter exceeding 50 nm. Using biotinylated USPIO particles it was possible to follow the accumulation of avidin-displaying viruses in choroid plexus cells in rat brain for up to 14 days (Figure 3).12Raty JK Liimatainen T Wirth T Airenne KJ Ihalainen TO Huhtala T et al.Magnetic resonance imaging of viral particle biodistribution in vivo.Gene Ther. 2006; 13: 1440-1446Crossref PubMed Scopus (48) Google Scholar The smallness of the USPIO particles allowed the viral particles to retain their Brownian diffusion and behave in a similar way to non-coated viruses in vivo. Similarly, successful MRI of the migration of stem cells in vivo has been achieved by feeding stem cells with SPIO particles in vitro.13Frank JA Anderson SA Kalsih H Jordan EK Lewis BK Yocum GT et al.Methods for magnetically labeling stem and other cells for detection by in vivo magnetic resonance imaging.Cytotherapy. 2004; 6: 621-625Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar,14Arbab AS Jordan EK Wilson LB Yocum GT Lewis BK Frank JA In vivo trafficking and targeted delivery of magnetically labeled stem cells.Hum Gene Ther. 2004; 15: 351-360Crossref PubMed Scopus (150) Google Scholar,15Modo M Hoehn M Bulte JW Cellular MR imaging.Mol Imaging. 2005; 4: 143-164PubMed Google Scholar SPIO- and USPIO-labeled therapeutic Schwann cells and olfactory ensheathing cells have been successfully imaged by MRI after transplantation into demyelinated lesions in rat central nervous system and rat spinal cord.16Dunning MD Lakatos A Loizou L Kettunen M Ffrench-Constant C Brindle KM et al.Superparamagnetic iron oxide-labeled Schwann cells and olfactory ensheathing cells can be traced in vivo by magnetic resonance imaging and retain functional properties after transplantation into the CNS.J Neurosci. 2004; 24: 9799-9810Crossref PubMed Scopus (131) Google Scholar,17Dunning MD Kettunen MI Ffrench Constant C Franklin RJ Brindle KM Magnetic resonance imaging of functional Schwann cell transplants labelled with magnetic microspheres.Neuroimage. 2006; 31: 172-180Crossref PubMed Scopus (30) Google Scholar More recently, USPIO-labeled activated T lymphocytes have been imaged in tumors undergoing immune rejection.18Hu DE Kettunen MI Brindle KM Monitoring T-lymphocyte trafficking in tumors undergoing immune rejection.Magn Reson Med. 2005; 54: 1473-1479Crossref PubMed Scopus (33) Google Scholar A recent study determined the threshold of cellular tracking with 30 pg of Fe/cell to be as low as 1,000 cells/mm3 with 3 T MRI and 500 cells/mm3 with 7 T MR.19Verdijk P Scheenen TW Lesterhuis WJ Gambarota G Veltien AA Walczak P et al.Sensitivity of magnetic resonance imaging of dendritic cells for in vivo tracking of cellular cancer vaccines.Int J Cancer. 2006; 120: 978-984Crossref Scopus (81) Google Scholar Altogether, these studies demonstrate that MRI has a clear advantage in stem cell–based therapies that require long-term in vivo tracking of specific cell populations. The MR contrasts generated by SPIO and USPIO particles are usually negative. However, with frequency-selective off-resonance excitation/refocusing in commonly used imaging sequences, a positive contrast can be achieved with paramagnetic markers20Seppenwoolde JH Viergever MA Bakker CJ Passive tracking exploiting local signal conservation: the white marker phenomenon.Magn Reson Med. 2003; 50: 784-790Crossref PubMed Scopus (170) Google Scholar and with SPIO-labeled cellsin vitro21Cunningham CH Arai T Yang PC McConnell MV Pauly JM Conolly SM Positive contrast magnetic resonance imaging of cells labeled with magnetic nanoparticles.Magn Reson Med. 2005; 53: 999-1005Crossref PubMed Scopus (379) Google Scholar and in vivo.22Foltz WD Cunningham CH Mutsaers AJ Conolly SM Stewart DJ Dick AJ Positive-contrast imaging in the rabbit hind-limb of transplanted cells bearing endocytosed superparamagnetic beads.J Cardiovasc Magn Reson. 2006; 8: 817-823Crossref PubMed Scopus (18) Google Scholar Positive contrast has the clear advantage in gene therapy applications that the underlying anatomical features are better visualized. Without the need for in vitrolabeling, cell surface receptors can be used to increase the concentration of contrast agents selectively. The expression of an engineered transferrin receptor23Moore A Josephson L Bhorade RM Basilion JP Weissleder R Human transferrin receptor gene as a marker gene for MR imaging.Radiology. 2001; 221: 244-250Crossref PubMed Scopus (148) Google Scholar has been imaged using an SPIO probe with a herpes simplex virus (HSV)–based amplicon vector system.24Ichikawa T Hogemann D Saeki Y Tyminski E Terada K Weissleder R et al.MRI of transgene expression: correlation to therapeutic gene expression.Neoplasia. 2002; 4: 523-530Abstract Full Text PDF PubMed Scopus (111) Google Scholar This is a good example of the ability of MRI to image gene expression in cells overexpressing receptors. However, the local concentration of the contrast agent needs to be approximately 10–3–10–5 mol/l,4Massoud TF Gambhir SS Molecular imaging in living subjects: seeing fundamental biological processes in a new light.Genes Dev. 2003; 17: 545-580Crossref PubMed Scopus (1848) Google Scholar which is substantially higher than the concentration of 10–11–10–12 mol/l required for PET detection. For in vivo imaging, the mass of contrast agent required could be problematic. A recently described method that bypasses the administration of the contrast agent by using native ferritin receptors offers an elegant way of concentrating the body's natural iron to quantities detectable by MRI. This method was used to image viral transduction in rat brain in a non-invasive manner without any external ligands. Genove et al. used adenoviruses to deliver a ferritin transgene into specific host tissues and imaged cells that took up endogenous iron and became superparamagnetic.25Genove G Demarco U Xu H Goins WF Ahrens ET A new transgene reporter for in vivo magnetic resonance imaging.Nat Med. 2005; 11: 450-454Crossref PubMed Scopus (368) Google Scholar In this context, the overexpressed ferritin receptors have been imaged using a heavy chain of murine ferritin, an iron storage molecule with ferrioxidase activity, for the detection of gene expression by MRI in vivo.26Cohen B Dafni H Meir G Harmelin A Neeman M Ferritin as an endogenous MRI reporter for noninvasive imaging of gene expression in C6 glioma tumors.Neoplasia. 2005; 7: 109-117Abstract Full Text PDF PubMed Scopus (279) Google Scholar Paramagnetic chelates that change their magnetic properties at enzymatic hydrolysis ("smart" contrast agents) have also been used for gene expression imaging.27Delikatny EJ Poptani H MR techniques for in vivo molecular and cellular imaging.Radiol Clin North Am. 2005; 43: 205-220Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,28Bell JD Taylor-Robinson SD Assessing gene expression in vivo: magnetic resonance imaging and spectroscopy.Gene Ther. 2000; 7: 1259-1264Crossref PubMed Scopus (36) Google Scholar In one approach, the enzymatic activity of β-galactosidase was utilized to remove a blocking group of galactopyranose and alter the contrast properties of the probe.11Louie AY Huber MM Ahrens ET Rothbacher U Moats R Jacobs RE et al.In vivo visualization of gene expression using magnetic resonance imaging.Nat Biotechnol. 2000; 18: 321-325Crossref PubMed Scopus (983) Google Scholar In general, the limited sensitivity of MRI restricts its use in molecular imaging applications. Nevertheless, several approaches combining targeted and/or smart contrast agents with biological amplification strategies have shown the feasibility of using MRI in gene transfer applications. PET probes are labeled with a positron-emitting isotope. The positron emitted from the isotope's nucleus annihilates with a nearby electron to produce two 511-keV γ-rays approximately 180° apart. Positron-emitting isotopes in common use include 15O, 13N, 11C, and 18F as a substitute for hydrogen.29Walker RC Purnell GL Jones-Jackson LB Thomas KL Brito JA Ferris EJ Introduction to PET imaging with emphasis on biomedical research.Neurotoxicology. 2004; 25: 533-542Crossref PubMed Scopus (19) Google Scholar A PET system usually includes a circle of detectors surrounding the imaging target. Because simultaneous γ-quanta from the annihilation event are detected, collimators are not required. This means that PET has greater sensitivity than SPECT. However, the radionuclides used in PET have very short half-lives (e.g., 11C T0.5 = 20.3 minutes), and it is necessary to have a cyclotron near the PET scanner to produce the isotopes used for imaging, which increases the cost of PET imaging. Because calculation of the origin of decay is simple, the sensitivity and spatial resolution are higher for PET imaging than for SPECT imaging. SPECT is based on the detection of γ-quanta emitted by radionuclides injected into a target. With SPECT, the spatial variation of radio decay can be detected and a 3D map of radioactivity can be constructed from projection images collected by γ-cameras rotating around the subject that detect γ-emitting isotopes such as 99mTc, 111In, 123I, 125I, and 131I.30Rosenthal MS Cullom J Hawkins W Moore SC Tsui BM Yester M Quantitative SPECT imaging: a review and recommendations by the Focus Committee of the Society of Nuclear Medicine Computer and Instrumentation Council.J Nucl Med. 1995; 36: 1489-1513PubMed Google Scholar The isotopes used in SPECT detection have half-lives ranging from hours to days (e.g., 99mTc T0.5 = 6 hours and 111In T0.5 = 2.8 days). The half-life of SPECT isotopes enables easy delivery, which decreases the cost of imaging. PET imaging in gene therapy. Because of its high sensitivity, PET is widely used for receptor imaging, particularly for the central nervous system.31Heiss WD Herholz K Brain receptor imaging.J Nucl Med. 2006; 47: 302-312PubMed Google Scholar PET can also be used to monitor the efficacy of gene therapy vectors. For example, PET has been used to determine the effect of hyperthermia on adenoviral gene delivery.32Li GC He F Ling CC Hyperthermia and gene therapy: potential use of microPET imaging.Int J Hyperthermia. 2006; 22: 215-221Crossref PubMed Scopus (12) Google Scholar Reporter gene imaging can provide quantitative transduction data for viral vectors, spatial data about the transduction pattern and persistence of the vector used, and even data on endogenous molecular events in various reporter gene systems.33Serganova I Blasberg R Reporter gene imaging: potential impact on therapy.Nucl Med Biol. 2005; 32: 763-780Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 34Penuelas I Boan J Marti-Climent JM Sangro B Mazzolini G Prieto J et al.Positron emission tomography and gene therapy: basic concepts and experimental approaches for in vivo gene expression imaging.Mol Imaging Biol. 2004; 6: 225-238Crossref PubMed Scopus (35) Google Scholar Among the many reporter genes suitable for PET imaging (reviewed in ref. 35Herschman HR PET reporter genes for noninvasive imaging of gene therapy, cell tracking and transgenic analysis.Crit Rev Oncol Hematol. 2004; 51: 191-204Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), the thymidine kinase transgene from HSV (HSV-tk)36Tjuvajev JG Avril N Oku T Sasajima T Miyagawa T Joshi R et al.Imaging herpes virus thymidine kinase gene transfer and expression by positron emission tomography.Cancer Res. 1998; 58: 4333-4341PubMed Google Scholar and its mutant version HSV-sr39tk37Gambhir SS Bauer E Black ME Liang Q Kokoris MS Barrio JR 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. 2000; 97: 2785-2790Crossref PubMed Scopus (400) Google Scholar have been used extensively. When HSV-tk is present in cells, the substrate is phosphorylated by the viral thymidine kinase and thus "trapped" in the radiolabeled cells. Radioactivity is therefore an indicator of thymidine kinase activity and thus of transduction of the tk gene. Unlike human thymidine kinases, viral enzymes have less substrate specificity and can selectively phosphorylate a wide range of compounds. HSV-tk, which is also used in anti-cancer treatments to promote apoptosis, has been successfully imaged with different radionuclides, including iodinated or fluorinated acycloguanosines (e.g., ganciclovir, penciclovir, 18F-labeled 9-[4-fluoro-(hydroxymethyl)butyl]guanine)38Gambhir SS Barrio JR Phelps ME Iyer M Namavari M Satyamurthy N et al.Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography.Proc Natl Acad Sci USA. 1999; 96: 2333-2338Crossref PubMed Scopus (402) Google Scholar,39Namavari M Barrio JR Toyokuni T Gambhir SS Cherry SR Herschman HR et al.Synthesis of 8-[(18)F]fluoroguanine derivatives: in vivo probes for imaging gene expression with positron emission tomography.Nucl Med Biol. 2000; 27: 157-162Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar,40Jacobs A Voges J Reszka R Lercher M Gossmann A Kracht L et al.Positron-emission tomography of vector-mediated gene expression in gene therapy for gliomas.Lancet. 2001; 358: 727-729Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar and thymidine analogues (e.g., 2′-fluoro-2′-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil and 2′-[18F]fluoro-5-ethyl-1-beta-D-arabinofuranosyl-uracil).41Yaghoubi S Barrio JR Dahlbom M Iyer M Namavari M Satyamurthy N et al.Human pharmacokinetic and dosimetry studies of [(18)F]FHBG: a reporter probe for imaging herpes simplex virus type-1 thymidine kinase reporter gene expression.J Nucl Med. 2001; 42: 1225-1234PubMed Google Scholar,42Buursma AR Rutgers V Hospers GA Mulder NH Vaalburg W de Vries EF 18F-FEAU as a radiotracer for herpes simplex virus thymidine kinase gene expression: in-vitro comparison with other PET tracers.Nucl Med Commun. 2006; 27: 25-30Crossref PubMed Scopus (55) Google Scholar These imaging tracers freely cross the plasma membrane by means of an active transport mechanism. With viral imaging, lentivirus transduction in vivo has been followed using 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-76Br-5-bromouracil (76Br-FBAU) as the imaging agent. The 16.2-hour half-life of 76Br-FBAU facilitated imaging in pharmacokinetic studies and supported the use of PET in gene therapy imaging.43Sander WE Metzger ME Morizono K Bonifacino A Penzak SR Xie YM et al.Noninvasive molecular imaging to detect transgene expression of lentiviral vector in nonhuman primates.J Nucl Med. 2006; 47: 1212-1219PubMed Google Scholar Among the many available radioprobes, 18F-FHBG has been used as an effective substrate for HSV1-tk and evaluated as superior to other agents in many imaging applications (e.g., in the study of breast cancer).44Alauddin MM Shahinian A Gordon EM Conti PS 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. 2004; 3: 76-84Crossref PubMed Scopus (55) Google Scholar In liver cancer patients, PET imaging showed that adenovirus-mediated HSV-tk expression disappeared within 9 days in all patients, aiding the assessment of clinical efficacy.45Penuelas I Mazzolini G Boan JF Sangro B Marti-Climent J Ruiz M et al.Positron emission tomography imaging of adenoviral-mediated transgene expression in liver cancer patients.Gastroenterology. 2005; 128: 1787-1795Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar The use of extracellular receptors or cell membrane transporters as reporter genes eliminates the need for the probe to penetrate into cells. The human dopamine receptor (human D2R) is one such reporter gene. Human D2R expression is largely limited to the striatal-nigral system of the brain, which makes it a good candidate for transduction imaging. D2R-expressing cells and tissues can be imaged through the accumulation of probes developed for each receptor subtype.46Elsinga PH Hatano K Ishiwata K PET tracers for imaging of the dopaminergic system.Curr Med Chem. 2006; 13: 2139-2153Crossref PubMed Scopus (113) Google Scholar A norepinephrine transporter–based reporter system has been used to image transduced tumors.47Altmann A Kissel M Zitzmann S Kubler W Mahmut M Peschke P et al.Increased MIBG uptake after transfer of the human nore