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Figure 8 Continued.

regarding such indirect approaches are provided in a later section.

In radionuclide imaging, one way to perform indirect imaging is to couple the therapeutic gene to a reporter gene that encodes a receptor that can bind, and, therefore, trap radiolabeled ligands. Following transfection, targeted cells may express the therapeutic gene product and receptor in proportional amounts. Subsequently, the ectopically expressed receptor will either localize to the cell membrane and/or remain intra-cellular. Following exposure to radiolabeled ligand probe, cells expressing the receptor will specifically bind the probe, resulting in a complex that can be detected by PET/SPECT/ gamma camera imaging. The intensity of activity on the PET image is directly proportional to the number of these receptor-ligand complexes, and, therefore, correlated to the amount of therapeutic gene expressed. Ideally, those cells not producing the receptor will be devoid of tracer signal.

An example of a receptor-based reporter gene is the dopamine type 2 receptor reporter gene (D2R) (49). D2R is normally an endogenous, cell-surface receptor predominantly expressed in the striatum. When activated, it causes a G-protein-coupled reduction of cyclic adenosine monophosphate (cAMP) via its inhibition of adenylate cyclase. When D2R is used as a reporter gene, a radiolabeled D2R antagonist, spiperone [3-(2' [18F]fluoroethyl)spiperone ([18F]FESP)], serves as the receptor's ligand and accumulates intracellularly and on the cell surface of D2R-expressing tissue. Radiolabeled spip-

erone, originally used to monitor levels of endogenous levels of striatal D2 receptors in vivo, binds to D2R with high affinity and is able to cross the blood-brain barrier. To overcome potential deleterious effects of ectopic D2R activation by circulating endogenous ligands, mutant D2R reporter genes, D2R80A and D2R194A, have been created that are disengaged from downstream transduction events while maintaining a high binding affinity for ligand probe (48).

A few advantages of the D2R-based reporter system are worth commenting. [18F]FESP's ability to cross the BBB and cell membranes favors its use in the central nervous system relative to other reporter systems such as the HSV1-tfc (mutant or not) reporter systems since [18F]FHBG is not as efficient in crossing this important barrier. Furthermore, [18F]FESP has a relatively easier time of localizing to target, which is a cell-surface and intracellular receptor. In contrast, [18F]FHBG has to cross the cell membrane in order to interact with the target enzyme and is, therefore, subject to transport kinetics. Also, D2R, an endogenous protein, is not immunogenic compared to HSV1-TK and therefore probably more appropriate for repeated imaging during longitudinal studies. Interestingly, despite these relative advantages of the D2R/[18F]FESP system, equivalent sensitivities are reported between the D2R/ [18F]FESP and mutant HSV1-tfc/[18F]FHBG PET reporter gene imaging systems in the liver (~20% ID/g in the liver when used with adenoviral delivery systems carrying constitutive CMV-based expression of the reporter gene). One must

Figure 9 Gamma Camera Imaging of the Sodium Iodide Symporter Transgene Recently cloned rat and human sodium iodide symporter genes (rNIS and hNIS, respectively) are increasingly showing their potential as a novel suicide gene therapy for a variety of cancer models. Once transduced to a cancer cell line, tumor, or target organ via recombinant viral transfection or liposomal-mediated techniques, the expressed membrane proteins facilitate the active intracellular transport of iodide (I~) into targeted cells. Exogenously administered radiotracers 1231,125I, or 99mTc-pertechnetate results in intracellular accumulation of these tracers in cells that are transduced with this symporter. The distribution of these tracers can be imaged with a gamma camera or SPECT and, therefore, be used as a means of localizing cells that have been transduced with NIS. Similarly, targeted brachytherapy can be performed by the administration of 131I. (A) A retroviral vector carrying the rNIS gene was used to transduce A375 human melanoma cell line. Transduced (NIS) and nontransduced (NV) tumor xenografts were subcutaneously implanted into the left and right flank of the photographed mouse, respectively. (B) By 30 days, the tumor had reached approximately 10 mm in diameter. An intraperitoneal dose of 131I was administered, and a gamma camera image was obtained after a 1-h incubation period. rNIS-transduced xenograft (left flank) demonstrates radioiodide uptake while nontransduced tumor (right flank) does not. The thyroid, stomach, and, to a lesser extent, the salivary glands endogenously express sodium iodide symporters and, thus, normal, physiological radioiodide uptake is seen in these organs. (Images reproduced with permission from Ref. 63.)

Figure 9 Gamma Camera Imaging of the Sodium Iodide Symporter Transgene Recently cloned rat and human sodium iodide symporter genes (rNIS and hNIS, respectively) are increasingly showing their potential as a novel suicide gene therapy for a variety of cancer models. Once transduced to a cancer cell line, tumor, or target organ via recombinant viral transfection or liposomal-mediated techniques, the expressed membrane proteins facilitate the active intracellular transport of iodide (I~) into targeted cells. Exogenously administered radiotracers 1231,125I, or 99mTc-pertechnetate results in intracellular accumulation of these tracers in cells that are transduced with this symporter. The distribution of these tracers can be imaged with a gamma camera or SPECT and, therefore, be used as a means of localizing cells that have been transduced with NIS. Similarly, targeted brachytherapy can be performed by the administration of 131I. (A) A retroviral vector carrying the rNIS gene was used to transduce A375 human melanoma cell line. Transduced (NIS) and nontransduced (NV) tumor xenografts were subcutaneously implanted into the left and right flank of the photographed mouse, respectively. (B) By 30 days, the tumor had reached approximately 10 mm in diameter. An intraperitoneal dose of 131I was administered, and a gamma camera image was obtained after a 1-h incubation period. rNIS-transduced xenograft (left flank) demonstrates radioiodide uptake while nontransduced tumor (right flank) does not. The thyroid, stomach, and, to a lesser extent, the salivary glands endogenously express sodium iodide symporters and, thus, normal, physiological radioiodide uptake is seen in these organs. (Images reproduced with permission from Ref. 63.)

also note that endogenous D2R expression in the striatum will produce background noise and, therefore, [18F]FESP imaging will only be useful outside the striatum.

Another receptor-based reporter system takes advantage of the somatostatin membrane receptors (SSTR), which also belong to the family of G-protein-coupled receptors. Under normal conditions, the interaction between SSTR and its li-gand, somatostatin (SS), is known to have a variety of biological effects including a role in vasoconstriction, immunomodulation, and an inhibitory effect on endocrine and exocrine secretory functions; more recently, SSTR-activated signal transduction pathways have been implicated in the induction of apoptosis and inhibition of cell growth (69) (70). SSTRs have also been found in normal and hyperplastic human endo-thelium where it is felt that the SSTR exerts negative effects on angiogenesis. Five different somatostatin receptor subtypes have been described thus far (SSTR1 through SSTR5), and their respective genes have been cloned (71).

Historically, the development of this reporter gene centered around probes that were already in existence and were being used clinically to identify diseased states characterized by upregulated SSTR receptors. Radiolabeled somatostatin analogs, for example, have been clinically used to identify a number of primary human cancers and their metastases where elevated levels of somatostatin receptors are seen. Neuroendocrine tumors (including carcinoid, islet cell tumors, small-cell lung cancers, pheocromocytomas, gastrinoma, par-agangliomas, and medullary thyroid cancers), pituitary gland tumors as well as sarcomas, meningiomas, low-grade astrocto-mas, lymphomas, some breast cancers, and metastatic prostate cancers are known to express high levels of SSTR, particularly SSTR2. In fact, the combination of high SSTR receptor den sity seen in some tumors and the antiproliferative, antian-giogenic, and antisecretory effects of SS analogs form the premise for somatostatin analog therapy for cancer patients (72,73).

The favorable binding kinetics of SS-SSTR are the basis for use of the sstr2 gene as reporter gene. Currently, [111In]-DTPA-D-Phe1-octreotide (Octreoscan) and [99mTc]-depreo-tide (Neotect) are radiolabeled somatostatin analogs that have been in routine clinical use for the past several years for the imaging of SSTR-positive tumors using gamma cameras or SPECT, i.e., SSTR scintigraphy (74,75). These agents have also been useful in the imaging of non-neoplastic conditions that are associated with SSTR upregulation, such as a variety of autoimmune and granulomatous diseases.

The potential of using sstr2 as a reporter gene has been realized. Fig. 10 demonstrates adenoviral delivery via intratu-moral injection of human sstr2 gene into a tumor xenograft of a mouse. The transfected tumor is subsequently detected with a gamma camera following an intravenous injection of [99mTc]P2045, a somatostatin analog (76). For sstr2 to serve as a true reporter gene, however, it will have to be uncoupled from signal transduction since it may have undesireable effects in targeted and surrounding cells in its current state.

On a side note, sstr2 is being tried as a ''double-edged'' suicide transgene for cancer therapy. Not only does SSTR primarily mediate antimetabolic effects as described earlier, but it can also be utilized for its ability to internalize and retain SS analogs. Efforts are being made to deliver cytotoxic agents to targeted cells by using the receptor as a courier of toxic SS analogs, such as [90Y]-DOTA-D-Phe1-Tyr3-octreotide ([90Y]-SMT 487). 90Y exerts its lethal effects by local irradiation via emitted p particle. When SSTR-null tumor xenografts are injected with recombinant adenovirus encoding the SSTR2 receptor, they become susceptible to systemically administered [90Y]-SMT 487 (77). This version of targeted radiotherapy is able to significantly reduce quadrupling times of the xenograft. When the sstr2 transgene is used in this manner it can be directly imaged using Octreoscan.

Indirect imaging can also be accomplished by coupling the therapeutic gene to a reporter gene that encodes an enzyme which converts freely dispersible radiolabeled substrate probes into sequestered products (78). For example, previously described HSV1-ifc or HSV1-sr39ifc can be used as a reporter gene by using [124I]FIAU or [18F]FHBG at tracer levels (subpharmacological, nontherapeutic dose). In this manner, the HSV1-ifc or mutant counterpart can be utilized strictly as a reporter gene.

C. MRI Reporter Genes

Imaging transgene expression using MRI depends on reporter genes that encode receptors or enzymes which specifically interact with probes which are attached or chemically modified to accommodate paramagnetic or supraparamagnetic substances. Once localized to their targets, these probes alter the local magnetic field, which changes the relaxivity of nearby protons, and, concomitantly, effects a change in the radiofre-

quency signal detected by the receiver. At least two MRI reporter systems have been proven in animal studies: engineered transferrin receptor (ETR)-dependent reporter systems and the ZacZ-EgadMe system.

Iron (Fe) is a superparamagnetic ion that can cause significant changes of the local magnetic field, which can be detected by MRI if supraphysiological concentrations can be achieved. Transferrin (Tf), an iron-binding protein, and its cell-surface receptor (Tf-R), a receptor ubiquitously present on most cell types, mediate normal cellular iron metabolism and regulation. Normal intracellular stores of iron are dependent upon internalization kinetics of this receptor-ligand complex. By removing the 3' untranslated region (UTR) regulatory sequence from the Tf-R gene and mRNA destabilization motifs in the 3' untranslated region, engineered transferrin receptor (ETR) has been created to be constitutively overexpressed and liberated from feedback regulatory control (79,80). As expected, ETR-transfected cells accumulate approximately 500% more probe (holo-Tf) than control cells. To further augment the difference between transfected and control cells, the reporter probe itself has been modified to possess even greater magnetic susceptibility characteristics. It involves the synthesis of a 3 nmmono-crystalline iron oxide nanoparticle (MION) that is surrounded by a layer of low molecular-weight dextran to which holotran-sferrin is covalently bound (Tf-MION). On the average, each MION particle contains approximately 2000 superparamag-netic Fe atoms (compared with the 2 Fe atoms present in a single molecule of the paramagnetic chelate, holotransferrin). As expected, T2-weighted gradient echo MR imaging (1.5T; imaging time 3-7 min per sequence; voxel resolution 300 x 300 x 700 ^m) reveals significantly lower signal intensity in ETR + tumor xenografts than control following intravenous administration of Tf-MION (79). Recently, a second superpar-amagnetic reporter probe, a dextran cross-linked iron-oxide (CLIO) superparamagnetic particle conjugated to trasnferrin (Tf-CLIO) has also been effective in identifying ETR + tissues with MR imaging (81).

The other MR imaging method shown to be compatible with living subjects relies on an enzymatic amplification strategy to monitor gene expression. As mentioned before, gadolinium (Gd) is a rare-earth element with the largest number of unpaired electrons. With 7 unpaired electrons, Gd is a strong paramagnetic substance, collectively affecting the spins of water protons immediately surrounding it. The increased signal (for T1-weighted sequences) seen in Gd-enhanced MR imaging is afforded by the increased relaxation rate of intimately associated water protons surrounding a Gd atom. Gadolinium's ability to affect the relaxation rate of protons varies inversely to the distance between the paramagnetic ion and water protons.

Based on these principles, a reporter probe, (1-(2-(p-galac-topyranosyloxy)propyl)-4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane)gadolinium(III) (EgadMe), has been cleverly formed by ''encasing'' Gd in a water-proof package—an artificial barrier or cage designed to keep water molecules at bay so as not to be affected by the Gd's magnetic personality (Fig. 11A) (82). In this configuration, water has

Figure 10 Gamma Camera Imaging of a Dual Promoter Construct (A) To measure target (therapeutic) gene expression, several strategies are employed to "link" the therapeutic gene with a reporter gene on a single vector. In one strategy, indirect measurements of a target gene expression can be made by a downstream reporter gene with both genes driven by separate and identical promoters (i.e., the 'dual-promoter' construct). Replication-incompetent adenovirus encoding human type 2 somatostatin receptor (hsstr2) and the herpes simplex thymidine kinase (TK) enzyme (Ad-CMV-hsstr2-CMV-tk) is an example of this construct. Both transgenes are driven by the cytomegalovirus promoter element. (B) Human nonsmall cell lung cancer tumor xenografts were subcutaneously implanted in the right and left flank of a mouse. The left tumor was injected with Ad-CMV-tk and the right tumor was injected with Ad-CMV-hsstr2-CMV-tk. Forty-eight h later, the mouse was simultaneously injected (IV) with both 99mTc-P2045, which is a somatostatin receptor peptide ligand, to detect expression of hsstr2 and radioiodinated 131I-2'-deoxy-2'-fluoro-P-D-arabinofuranosyl-5-iodouracil (FIAU) to detect TK expression. Imaging was performed with an Anger gamma camera 5 h after injection of the radiotracers. The gamma camera can be 'tuned' to select for gamma rays that fall within a defined range. It is this property of the gamma camera that can discriminate among the activity of different radioisotopes such as 99mTc (140 keV) and 131I (364 keV). Thus, 2 images can be obtained from the same animal by changing the window settings in the gamma camera—one for 99mTc (for hsstr2 expression, lower left image) and the other for 131I (for TK expression, lower right image). The right flank xenograft, which was injected with the dual promoter construct (Ad-CMV-hsstr2-CMV-tk), demonstrates uptake of both radiotracers, while the left flank xenograft (injected with Ad-CMV-tk) demonstrates [131I]FIAU uptake only. These finding support the feasibility of the dual promoter approach for tracking transgene delivery. (Images reproduced with permission from Ref. 76.)

Figure 11 Magnetic Resonance Imaging (MRI) of P-galactosidase-activated MRI Reporter Probe (EgadME) One can obtain image contrast in MRI by using paramagnetic substances that change the local magnetic field and, thereby, increase the relaxation rate of nearby water protons. Gadolinium (Gd) is an example of a paramagnetic substance, and a relatively high local concentration of this agent translates into enhanced brightness as seen on T1-weighted images. (A) A reporter probe, EgadMe, has been formed by 'encasing' Gd in a molecular casing—an artificial barrier designed to keep water molecules at bay so as not to be affected by the Gd's magnetic effects. In this configuration, water has no access to the paramagnetic ion, and therefore, this probe is 'silent' on MR imaging. Part of the physical barrier is composed of a sugar, a galactopyranose cap, which has been attached to the cage by a p-galactosidase-cleavable linker. If lacZ is used as a reporter gene, subsequent enzyme cleavage releases the cap and allows water access to the gadolinium ion, thus, 'activating' this novel MR contrast agent. (B) EgadMe permits MRI detection of lacZ gene expression. Linearized plasmid cDNA encoding lacZ is injected into 1 of the cells of the 2-cell stage Xenopus laevis embryo. EgadMe is injected into both cells of the 2-cell stage. Subsequent enzyme expression is on 1 side of the animal since the 2 cells represent the future right and left sides of the animal. MR imaging of the embryos has been obtained at approximately the 100,000-cell stage using a 11.7 T magnet. As expected, p-galactosidase activity is seen in one half of the animal depicted as areas of high signal intensity within the endoderm (e) and head (h). (C) Light microscopic images of the same embryo fixed and stained with X-gal. Areas of X-gal staining follow regions of high signal intensity on MR image. (Images reproduced with permission from Ref. 82.)

no access to the paramagnetic ion and is therefore this probe is ''silent'' on MR imaging. As cleverly designed, part of the physical barrier is composed of a sugar—a galactopyranose cap, which has been attached to the cage by a p-galactosidase-cleavable linker. If lacZ is used as a reporter gene, subsequent enzyme cleavage releases the cap and allows water access to the gadolinium ion, thus, ''activating'' this novel MR contrast agent. p-galactosidase activity, following introduction of linearized plasmid cDNA encoding lacZ into a specific subset of cells in a Xenopus laevis embryo, has been imaged after an intracellular injection of EgadMe (Fig. 11B,C).

In its current state, EgadMe has difficulty crossing cell membranes and, as a result has to be directly injected intracel-lularly to maximize detection in vivo. Furthermore, relatively slow kinetics of cleavage for this agent is perhaps suboptimal for imaging gene expression (82). Regardless, these relatively recent developments will be refined, and they indicate great potential for MR imaging of transgene expression.

D. Magnetic Resonance Spectroscopy (MRS) Reporter Genes

Certain metabolites produced physiologically from endogenous enzymes or uniquely from exogenous enzymes have unique chemical signatures that can be detected using MRS (a.k.a., nuclear magnetic resonance (NMR) spectroscopy). These enzymes can be overexpressed in target tissues and can be used as MRS reporter genes to successfully track gene expression in transgenic models, transfected tumor xenografts, or viral-mediated gene transfer experiments. At this time, MRS does not produce true, spatial ''pictures'', and, instead, shows spectral tracings of the various metabolites it is able to identify. It is a sensitive, quantitative, and relatively fast technique when compared to MRI (29).

One particular MRS-sensitive metabolic reaction produces ATP, a process catalyzed by creatine kinase (CK):

where PCr is phosphocreatine and Cr is creatine. More specifically, 31P-MRS identifies amounts of phosphocreatine (PCr), ATP, ADP, and free phosphorus in the reaction. This molecule can be detected readily in the heart, muscle, and brain since they are produced in great quantities in these organs. The liver, on the other hand, has very low levels and thus can serve as a background for situations where CK is overexpressed. A transgenic mouse model that overexpresses this enzyme in the liver has shown that it can generate MRS-detectable levels of PCr (83). This technique is however invasive in nature, for a tissue window has to be created to minimize background noise from the overlying muscle and other surrounding structures rich in CK. Future developments in 31P 3D spectroscopy may eventually prove helpful.

A related study employs an invertebrate analog of CK as its reporter gene. Drosophila melanogaster arginine kinase (AK) has been cloned and, when introduced into mammalian muscle, produces phosphoarginine (PArg) in the following reaction (84):

PArg provides a unique phosphorus signal that is amplified when the transgene for AK is delivered to mammalian tissues (Fig. 12). One important consideration in the use of Droso-phila AK is it can act as an ATP buffer in mammalian tissues and, thus, the consequences of this need to be explored prior to its widespread use as an in vivo gene reporter gene.

Another MRS-friendly system is the cytosine deaminase (CD) reporter system (85). Using 19F MRS, the conversion of the relatively benign 5-fluorocytosine (5-FC) to the cytotoxic agent (5-FU) driven by this enzyme can be detected. Tumor xenografts transfected to express yeast CD have been shown to produce 5-FU with MRS.

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