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Figure 6 Optical (Bioluminescence) Imaging of Cardiac Reporter Gene Expression Replication-defective adenovirus carrying firefly luciferase (Fluc) driven by a constitutive cytomegalovirus (CMV) promoter (Ad-CMV-Fluc, 1 x 109 pfu) was injected directly into the myocardium (anterolateral wall) of a rat. Images obtained 2, 5, 8, and 14 days later from a cooled CCD camera demonstrate significant cardiac emissions from firefly luciferase activity (P<0.05 vs. control). By day 8, luciferase activity is seen in the liver, which is probably from spillover of adenoviral vector into the systemic circulation and subsequent hepatic transfection of the virus via coxsackie-adenovirus receptors on hepatocytes. On day 14, the heart of the same rat was explanted after whole-body imaging was performed and sliced into 3 sections (bottom right). Firefly luciferase activity is localized at the anterolateral wall of the left ventricle along the site of virus injection. Control rats, which received an intracardiac injection of Ad-CMV-HSV1-sr39f£ (1 x 109 pfu), demonstrate no significant firefly luciferase activity 2 days after the injection (upper left). Please note the bioluminescent scales are different for control rat, study rat days 2 to 5, and study rat days 8 to 14 to account for the wide range of cardiac firefly luciferase activity observed. Scales are a quantitative indicator of light photons detected [relative light units (RLU)/minute (min)]. RA indicates right atrium; RV, right ventricle; and LV, left ventricle. See color insert for color version of this figure. (Images reproduced with permission, Ref. 116.)

STUDY STUDY EXPLANTED HEART RLU/mln (DAY 8) (DAY 14) (DAY 14)

Figure 6 Optical (Bioluminescence) Imaging of Cardiac Reporter Gene Expression Replication-defective adenovirus carrying firefly luciferase (Fluc) driven by a constitutive cytomegalovirus (CMV) promoter (Ad-CMV-Fluc, 1 x 109 pfu) was injected directly into the myocardium (anterolateral wall) of a rat. Images obtained 2, 5, 8, and 14 days later from a cooled CCD camera demonstrate significant cardiac emissions from firefly luciferase activity (P<0.05 vs. control). By day 8, luciferase activity is seen in the liver, which is probably from spillover of adenoviral vector into the systemic circulation and subsequent hepatic transfection of the virus via coxsackie-adenovirus receptors on hepatocytes. On day 14, the heart of the same rat was explanted after whole-body imaging was performed and sliced into 3 sections (bottom right). Firefly luciferase activity is localized at the anterolateral wall of the left ventricle along the site of virus injection. Control rats, which received an intracardiac injection of Ad-CMV-HSV1-sr39f£ (1 x 109 pfu), demonstrate no significant firefly luciferase activity 2 days after the injection (upper left). Please note the bioluminescent scales are different for control rat, study rat days 2 to 5, and study rat days 8 to 14 to account for the wide range of cardiac firefly luciferase activity observed. Scales are a quantitative indicator of light photons detected [relative light units (RLU)/minute (min)]. RA indicates right atrium; RV, right ventricle; and LV, left ventricle. See color insert for color version of this figure. (Images reproduced with permission, Ref. 116.)

probe intracellularly and/or on the surface of cells expressing these genes. Ideally, those cells lacking the transgene will be unable to trap the reporter probe. The amount of probe or tracer used is typically in the nanogram range and does not lead to any pharmacological effect (46). In contrast, bioluminescence strategies, NIRF and MRI, require mass amounts of probe (usually ^g-mg) and, as a result, may produce a pharmacological effect.

Radionuclide imaging has significant advantages in that it permits quantitative and repetitive imaging in the same subject over time (47-50). Perhaps the main drawback of radionu-clide-based techniques is that unsequestered probe circulates

Figure 7 Optical (Bioluminescence) Imaging of Targeted Transgene Expression Using a Tissue-Specific Promoter One way to target gene therapy is through the use of tissue-specific promoters. However, most tissue-specific promoters yield low levels of transcription. In this example, certain key regulatory elements of the promoter and enhancer of prostate-specific antigen (PSA) have been multimerized to yield a construct, PSE-BC, which is 20-fold more active than the native PSA promoter/enhancer. Following incorporation into an adenovirus vector (AdPSE-BC-luc) and subsequent intratumoral injection into a human prostate cancer xenograft model (LAPC series), firefly luciferase expression can be seen in the main tumor xenograft (left flank) as well as other extratumoral sites, such as the back and chest, in this male SCID mice 21 days after vector delivery. Detailed histological analysis of the xenograft and extratumoral sites demonstrates that firefly luciferase expression is restricted to the prostate tumor and prostate metastases, respectively. The metastases, in this case, are located in the spine and lung. By comparison, CCD imaging and histological analysis of xenografts injected with AdCMV-luc show markedly diminished expression of firefly luciferase in the xenograft and increased nonspecific expression in the liver at 21 days postinjection (figure not shown). Results from this study indicate that tissue-specific transgene expression is possible and that CCD imaging can be used to track firefly luciferase-marked tumor cells. Scale indicates the number of photons detected (RLU/min). See color insert for color version of this figure. (Images reproduced with permission, Ref. 117.)

in the enterohepatic system or is excreted in the kidneys. As a result, probe can collect in the gut, kidneys, urinary bladder, or gall bladder, making it difficult to specifically evaluate these organs. Discussed below are 2 fundamentally different approaches to radionuclide-based reporter imaging: direct and indirect imaging.

1. Direct Radionuclide Imaging: HSV1-TK, Mutant HSV1-TK, Sodium Iodide Symporter, and Cytosine Deaminase

One of the major advantages of using a radionuclide technique is that it allows the opportunity to directly image delivered therapeutic genes (i.e., ''direct imaging''). In direct imaging, there is no need to couple the therapeutic gene with an additional reporter gene since reporter probes already exist for the therapeutic gene. The herpes simplex virus type 1 thymidine kinase gene (HSV1-ife) is an exemplary model of direct imaging.

Mammalian thymidine kinases phosphorylate thymidine for its normal incorporation into DNA during replication. The HSV1 thymidine kinase gene (HSV1-ife), on the other hand, is normally employed as a suicide gene product for the therapy of cancer. The gene product, with its broad substrate specificity, is able to phosphorylate acycloguanosine, guanosine, and thymidine derivatives and subsequently trap these substrates intracellularly much more efficiently than endogenous thymi-dine kinase. Thus, HSV1-ifc acts as a suicide gene when delivered with a prodrug (such as acyclovir, ganciclovir, and pen-ciclovir) since high concentrations of phosphorylated nucleoside analogs cause premature chain termination by their inhibition DNA polymerase, which leads to cell death. Transfected cell populations particularly affected by this type of therapy are those that have a high mitotic index. When radiolabeled acycloguanosines (e.g., 9-[(3-[18F] fluoro-1-hy-droxy-2-propoxy)methyl]guanine ([18F]FHPG); 9-(4-[18F] fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG)), guanosines (e.g., fluorogangciclovir ([18F]FPGV); fluoropenciclovir ([18F]FPCV)), and thymidine derivatives (2'-[124I] fluoro-2'deoxy-1-^-D-arabinofuranosyl-5-iodouracil ([124I]FIAU)) are used in nonpharmacological (trace) doses, they can serve as PET reporter probes. Similarly, [131I]FIAU and [123/ 125I]FIAU can be used as gamma camera or SPECT reporter probes. The details of the synthesis and kinetics of these and other similar agents have been reviewed (51-56).

Endogenous thymidine kinase, HSV1-ifc, and a mutated form of HSV1-ifc, HSV1-sr39ifc, each demonstrate different substrate specificity that can be exploited for therapeutic and imaging purposes. For example, endogenous thymidine kinase demonstrates narrow substrate specificity and cannot efficiently phosphorylate radiolabeled prodrugs. While endogenous thymidine kinase can phosphorylate the prodrugs/probes to a minor degree, nontransfected cells cannot accumulate significant amounts of the radiolabeled prodrugs. Instead, these radiolabeled prodrugs preferentially localize to cells, tissues, or tumors that express the HSV1-ifc gene.

Comparison of the HSV1-ifc probes demonstrates that [124I]FIAU displays favorable pharmacokinetics (greater sen sitivity, better contrast, less background noise) and, thus, the best imaging potential (57). [124I]FIAU, in particular, may be advantageous over the other tracers in some cases because the half-life of 124I is 4.2 days, allowing for longer systemic clearance. On the other hand, this longer half-life may also prove problematic in cases when frequent repeat imaging is needed since a minimum 10 to 12 days between imaging sessions is needed to allow clearance of prior probe administration. Further testing has shown that [18F]FHPG and [18F]FHBG are inferior agents for HSV1-TK. This variability seen amongst HSV1-TK reporter probes is in part due to differences in biological half-life, stability, substrate competition, degree of nonspecific binding, specific retention, rates of cellular transport, method of transfer (viral-mediated vs. stable transfection), and routes of clearance (58).

Mutated versions of the suicide gene, HSV1-ifc, have been created to enhance its killing potential by increasing its ability to phosphorylate prodrugs (59). From a library of site-directed mutants, it has been determined that the product of mutant HSV1-sr39ifc suicide gene is more adept at phosphorylating ganciclovir and less efficient in phosphorylating endogenous thymidine when compared to wild-type HSV1-TK. In light of this, mutant kinase has been exploited as a reporter gene since radiolabeled reporter probes already exist and are identical to those used for the wild-type HSV1-TK. As expected, HSV1-sr39TK efficiently phosphorylates [18F]FGCV, [18F]FPCV, [18F]FHPG, and [18F]FHBG, with [18F]FHBG as the most effective substrate for the mutant thymidine kinase. The ability to trap the reporter probe is improved by at least a factor of 2.0-3.0 in mutant TK-expressing tumor xenografts when compared with wild-type TK. Significantly improved uptake of the reporter probe is seen in the liver following systemic delivery of a recombinant adenoviral vector carrying the mutated transgene, resulting in enhanced sensitivity for imaging this transgene in vivo when compared to wild-type HSV1-ifc (60). An example of the use of HSV1-sr39ifc as a potential reporter gene for cardiac gene therapy is provided (Fig. 8). As an aside, an example of an optical reporter for use in cardiac gene therapy (using a nearly identical adenoviral vector and the firefly luciferase reporter gene) is provided for side-by-side comparison to better demonstrate the spatial advantages of microPET imaging (Fig. 8).

Another therapeutic transgene that lends itself to direct imaging in living subjects is the sodium/iodide symporter (NIS) gene (Fig. 9). NIS is an intrinsic membrane protein that is responsible for translocating and concentrating iodide within thyroid follicular cells. In this normal, physiological situation, iodide is eventually used to make the thyroid hormones (61). Recent cloning of the symporter has permitted the investigation of its role as a suicide gene. Because the symporter can concentrate high intracellular levels of iodide (including radioiodide, 131I), targeted cells expressing the symporter can be killed in this form of targeted radiotherapy; by the accumulation of radioiodide, it is estimated that a dose of up to 50,000 cGy of ionizing radiation can be achieved in targeted cancer cells (62)! The lethal effects of the NIS have been cleverly demonstrated in vitro, in a variety of cell lines including melanoma, colon cancer, and ovarian carcinoma, and in a murine model of a transfected melanoma xenograft following intraperitoneal injection of 131I (63). Also, tissue-specific expression of NIS transgene is possible by fusing a prostate-specific antigen (PSA) promoter fragment with the NIS gene—the result being tissue-specific expression of NIS transgene and subsequent accelerated death of an androgen-dependent prostatic carcinoma xenograft after injection with a therapeutic dose of radioiodine (64).

Imaging of the NIS transgene is relatively straightforward since the therapeutic agent, 131I, can also be used as an imaging agent with a gamma camera or SPECT. Similarly, 123I or 125I, both of which are commercially available, can also be used as reporter probes. A significant advantage of this reporter system is that the reporter probe is relatively simple to produce and commercially available. Specialized radiochemistry, such as that required for HSV1-tfc reporter probes, is not needed here. Another significant advantage of this system is that this reporter system can readily be imaged with PET using 124I as the positron-emitting reporter probe, which already is available (65); the very same living subject carrying the NIS transgene can be imaged with either SPECT or PET depending upon the choice of reporter probe. The main confounding issue with using this reporter system, on the other hand, is that radioiodine will not only localize to target cells, but will also accumulate in normal tissues such as the thyroid, salivary glands, breast, and stomach, all of which express physiological levels of endogenous NIS. As it currently stands, it is also not clear if the NIS approach is as sensitive as the HSV1-tA/sr39tfc approaches.

The cytosine deaminase (CD) transgene, another suicide gene, encodes an enzyme that converts 5-fluorocytosine to the toxic 5-fluorouracil (5-FU). Efforts to image this transgene, which have been performed with radiolabeled fluorocytosine as a probe, have largely been hampered by suboptimal phar-macokinetics: poor tracer uptake and poor retention of the toxic metabolite (66). Extended periods of up to 48 h are required to see differential accumulation of tracer between transfected and control cells. While targeted delivery of CD remains a viable method of suicide gene therapy (67), alternative substrates for CD, which are rapidly transported, deami-nated, and trapped intracellularly, will have to be developed in order to use this transgene as a reporter gene.

2. Indirect Radionuclide Imaging: D2R, Mutant D2R, and Somatostatin-2 Receptor

In cases where a reporter probe does not already exist for a delivered transgene, a number of strategies exist for indirect transgene imaging. Indirect imaging involves the simultaneous coexpression of the therapeutic gene and reporter gene with both driven by the same or identical promoter. Because reporter gene expression can directly correlate with transgene expression, such approaches have the potential to give us valuable information on the quantity and localization of transgene expression (68). It should be noted, however, that it is not a given that they directly correlate; one can hope that they do but each approach/application has to be tested. Further details

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Figure 8 Micropet and Optical (Bioluminescence) Imaging of Cardiac Reporter Gene Delivery (A) Imaging cardiac gene expression using adenoviral-mediated mutant thymidine kinase (HSV1-sr39f£) as PET reporter gene and [18F]FHBG as PET reporter probe. Trapping of tracer occurs only in cells expressing the reporter gene. At day 4, whole-body microPET image of a rat shows focal cardiac [18F]FHBG activity at the site of intramyocardial Ad-CMV-HSV1-sr39f£ injection. Liver [18F]FHBG activity is also seen because of systemic adenoviral leakage with transduction of hepatocytes. Control rat injected with Ad-CMV-Fluc shows no [18F]FHBG activity in either cardiac of hepatic regions. Radiolabeled probe is always ''visible'' with radionuclide imaging regardless of whether it has localized to its target or not. As a result, radionuclide-based images will exhibit a certain degree of nonspecific tracer localization since the ''unbound'' reporter probe is metabolized through either the enterohepatic or urinary system or both. In this example, nonspecific reporter probe activity and gut and bladder activities are seen for both study and control rats because of route of [18F]FHBG clearance. (B) Tomographic views of cardiac microPET images. The [13N]NH3 (gray scale) images of perfusion are superimposed on [18F]FHBG images (color scale), demonstrating HSV1-sr39f£ reporter gene expression. [18F]FHBG activity is seen in the anterolateral wall for experimental rat compared with background signal in control rat. Perpendicular lines represent the axis for vertical and horizontal cuts. Color scale is expressed as % ID/g. (C) Comparison of typical images obtained with PET (left) and optical imaging (right). The optical method is more sensitive (at limited depths), easier to perform, and demonstrates minimal background noise. With PET, we can see that the transgene was delivered to the anterolateral aspect of the left ventricle. Such spatial resolution is not afforded by in vivo optical imaging at this time. See color insert for color version of this figure. (Images reproduced with permission from Refs. 116,118.)

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