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Published ahead of print on December 27, 2002, doi:10.1164/rccm.200210-1217OC
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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1257-1263, (2003)
© 2003 American Thoracic Society

Imaging Pulmonary Gene Expression with Positron Emission Tomography

Jean-Cristophe Richard, Zhaohui Zhou, Datta E. Ponde, Carmen S. Dence, Philip Factor, Paul N. Reynolds, Gary D. Luker, Vijay Sharma, Tom Ferkol, David Piwnica-Worms and Daniel P. Schuster

Washington University School of Medicine, St. Louis, Missouri; Evanston Northwestern Healthcare, Evanston; Northwestern University Medical School, Chicago, Illinois; Department of Medicine, Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, Alabama; and Royal Adelaide Hospital, Adelaide, South Australia

Correspondence and requests for reprints should be addressed to Daniel P. Schuster, M.D., University Box 8225, Washington University School of Medicine, 660 South Euclid, St. Louis, MO 63110. E-mail: schusted{at}msnotes.wustl.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We evaluated positron emission tomographic imaging of pulmonary transgene expression, using an enhanced mutant herpes simplex virus-1 thymidine kinase as the reporter gene, in the lungs of normal rats. Sixteen rats were studied 3 days after an intratracheal administration of 5 x 109 to 1 x 1011 viral particles of a replication-incompetent adenovirus containing a fusion gene of the mutant kinase and green fluorescent protein. Three rats infected with adenovirus containing no insert (null vector) served as control subjects. Images were obtained 1 hour after an intravenous injection of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine, an imaging substrate for the viral kinase. After euthanasia, tissue radioactivity was determined in a {gamma} counter, and thymidine kinase activity and green fluorescent protein levels were measured in lung tissue samples. Imaging and {gamma} counting radioactivity measurements were strongly and linearly correlated (r2 = 0.96, p < 0.001). Imaging detected thymidine kinase expression above background (null vector) in 15 of 16 rats, even at low viral doses that produced little to no measurable green fluorescent protein expression. Lung 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine uptake (as assessed by imaging) correlated with in vitro assays of both kinase activity (r2 = 0.48, p < 0.001) and fluorescent protein (r2 = 0.46, p < 0.001). We conclude that positron emission tomographic imaging is a sensitive and quantitative method for detecting pulmonary reporter gene expression noninvasively.

Key Words: positron emission tomography • rats • green fluorescent protein • reporter gene

Despite daunting challenges and setbacks, prospects remain strong that pulmonary gene therapy will eventually become part of the routine therapeutic armamentarium. Although gene therapy was initially proposed as a curative approach for inherited monogenic diseases such as cystic fibrosis and {alpha}-1-antitrypsin deficiency, recent experimental studies have shown that acute illnesses might also benefit from gene transfer therapy (1). For instance, a transgene encoding the heat shock protein-70 has been successfully transferred to rat lungs after the onset of experimental lung injury, significantly decreasing lung interstitial and alveolar edema, neutrophilic accumulation in the lungs, and mortality (2). Detecting gene expression in the lungs has been especially difficult, and progress in the field of gene therapy in general will ultimately depend on appropriate clinical methods to evaluate the distribution, magnitude, and timing of transgene expression. Although in situ hybridization techniques, immunohistochemical procedures, and several reporter genes can be used to monitor gene expression in tissue biopsy samples, these methods are invasive and unattractive as routine procedures for clinical evaluation.

Recent advances in imaging technologies make noninvasive visualization of in vivo reporter gene expression possible. Noninvasive real-time analysis of gene expression is possible using reporter genes with optical signatures (e.g., green fluorescent protein [gfp] [3], firefly luciferase [4], and bacterial luciferase). However, spatial resolution is poor, and their use at present is limited to small animals. Gene expression imaging with magnetic resonance has been described, but this approach is not suitable for lung studies, as the proton density of the lung parenchyma is too low to generate a magnetic resonance image (5). On the other hand, radiotracer imaging offers the possibility of monitoring the detailed location, magnitude, and persistence of reporter gene expression, even clinically. Among these methods, positron emission tomography (PET) imaging has the greatest sensitivity and isotropism, and its usefulness as a method for gene expression imaging has been further enhanced by the development of dedicated small-animal PET scanners (6).

Recent studies have shown that PET can be used to detect reporter proteins such as the herpes simplex virus type-1 thymidine kinase (HSV1-TK) in vivo (7, 8). Unlike mammalian thymidine kinase, this enzyme can efficiently phosphorylate nucleoside analogues (e.g., ganciclovir, penciclovir), as well as various radioactive derivatives such as (9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine; [18F]FHBG), which are then trapped and accumulate in cells expressing the viral kinase. Several mutant HSV1-TK enzymes (mHSV1-TK) (8, 9) have been developed to further improve the sensitivity of PET imaging to detect and quantify the cellular accumulation of these radioactive tracers. This study was performed to demonstrate that these techniques can be used to detect the expression of genes delivered to the lungs by viral vectors.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture Studies
HeLa cells were infected with various viral titers (1 x 107 to 1 x 109 viral particles [VPs]) of a human type 5 replication deficient (E1a/E3 deleted) adenovirus (Ad), containing a fusion gene of mHSV1-tk and gfp (8), driven by the cytomegalovirus promoter (Ad-CMV-mNLS-sr39tk-egfp). An Ad containing no insert (AdCMVnull) served as the control vector. The cells were harvested 48 hours later for quantitation of mHSV1-TK activity and GFP expression by ELISA.

Ad Infection Protocol
This study was approved by the Washington University School of Medicine's Animal Studies Committee and was performed on 19 Sprague-Dawley rats (mean weight ± SD, 280 ± 70 g). Sixteen rats were infected with various viral doses ranging from 5 x 109 to 1 x 1011 VPs of AdCMV-mNLS-sr39tk-egfp. A control group (n = 3) was infected with a sham vector (AdCMVnull). All Ad delivery to rat lungs was performed using a surfactant-based method (10, 11).

PET Studies
[18F]FHBG was synthesized as previously described (12). PET imaging was performed on a microPET-R4 camera (13, 14). Rats were anesthetized with isoflurane shortly before a bolus injection of 1.3 ± 0.2 µCi/g of [18F]FHBG into the tail vein. After 1 hour, each animal was placed in the supine position into the microPET scanner, with image acquisition over 30 minutes.

Image Analysis
Regions of interest were drawn on both lungs. All activity measurements (injected dose, PET images) were decay corrected to the same time point (beginning of the PET scan), and mean activity values per milliliter of lung for each region of interest were normalized to the injected dose of [18F]FHBG. Data from each of multiple image slices were divided into thirds, roughly corresponding to upper, middle, and lower lobes, respectively (Figure 1) .



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  		Figure 1. Method of region of interest placement on the PET images. The picture represents a coronal view of the lungs with PET after [18F]FHBG injection. All slices intersecting the right lung were divided into thirds, roughly corresponding to upper, middle, and lower lobes.

 
Tissue Retrieval Protocol
Immediately after PET scanning was completed, rats were killed. Lungs were harvested, immediately frozen, and later processed for mHSV1-TK enzyme activity (right middle lobe) and GFP levels by ELISA (right lower lobe). Radioactivity in the left lung was measured in a {gamma} counter, decay corrected to the beginning of the PET scan, and normalized to the [18F]FHBG injected dose. The result was expressed as the percentage of injected dose per gram of lung. After fixation, the left lung was cryosectioned and studied by fluorescence microscopy.

mHSV1-TK Enzyme Assay
Lung tissue extracts (1 µg of protein) were incubated for 20 minutes with [3H]-penciclovir to determine the formation of phosphorylated [3H]-penciclovir, which was subsequently trapped onto a DE-81 filter (Whatman, Maidstone, UK). Activity in the filter was measured with a ß counter. Results are reported as the percentage of phosphorylation of [3H]-penciclovir/µg protein/minute (15, 16).

GFP Quantitation with ELISA
Protein extracts were incubated in microplates coated with goat anti-GFP antibody, and bound protein was detected by horseradish peroxidase–conjugated anti-GFP antibody. The GFP levels were quantified using a spectrophotometer and were normalized by protein concentration of each sample.

Statistical Analysis
Regression analyses were performed by the least squares method, and correlations were assessed with Pearson's correlation coefficient. Comparisons among groups were performed by analysis of variance or analysis of variance on ranks. For additional pair-wise comparisons, Dunn's test was performed. The level of significance was set at p < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In vitro mHSV1-TK activity, measured in cell culture after transfection with Ad vector, showed a highly linear correlation with viral dose (Figure 2A) . GFP expression by ELISA showed a similarly strong correlation with viral dose (r2 = 0.997, data not shown). As a result, both measures of mHSV1-tk and gfp expression were also highly correlated to one another (Figure 2B). Comparable results were found when correlating measurements of mHSV1-TK activity and GFP expression from lung tissue samples obtained 3 days after viral vector administration to intact rats (Figure 3) . These data then support the validity of these gene expression assays and demonstrate that both components of the fusion protein were functional.



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  		Figure 2. mHSV1-tk and GFP expression levels in HeLa cells infected with various doses of AdCMV-mNLS-sr39tk-egfp. (A) In vitro correlation between viral dose and mHSV1-TK enzyme activity. (B) In vitro correlation between mHSV1-TK enzyme activity and GFP quantitation by ELISA. Note that the viral dose added to the cell culture in the AdCMVnull group was 1 x 109 VP. PCV = penciclovir.

 


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  		Figure 3. In vivo correlation between lung tissue mHSV1-TK activity and GFP quantitation by ELISA. Each point corresponds to a different rat (n = 16), and data from the Null vector rats have been excluded as they exhibit no GFP activity.

 
The validity of PET imaging as a means of measuring tissue radioactivity was demonstrated by showing that the lung uptake of [18F]FHBG (as assessed by PET analyses) was highly correlated to radioactivity measurements of ex vivo lung tissue samples (Figure 4) .



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  		Figure 4. Relationship between the PET signal and {gamma} counter activity in matching lung lobes. Each point corresponds to a different rat (n = 19). ID = injected dose.

 
Measurements of gene expression (evaluated by PET imaging, ex vivo mHSV1-TK enzyme activity, or ex vivo GFP levels) as a function of viral dose are shown in Figure 5 . For all viral doses, PET imaging detected lung mHSV1-tk expression above background (i.e., compared with measurements in rats administered null vector) (Figure 5A). However, with in vitro assessment of mHSV1-TK activity from lung tissue samples (Figure 5B) or quantitation of GFP by ELISA (Figure 5C), protein function was barely detectable at the lower doses but increased significantly at the higher doses. Likewise, little fluorescence was observed on histologic tissue sections taken from rats administered the lower doses of viral vector (Figure 6) . Thus, the in vitro assays appeared to display a higher detection threshold, requiring a viral dose of 5 x 1010 VP before the level of gene expression was sufficient to allow detection by these methods. In contrast, the detection threshold for PET imaging was lower but displayed a shallower dynamic response as the viral dose increased (Figures 5B and 5C vs. 5A).



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  		Figure 5. Quantitation of the PET signal (A), mHSV1-TK enzyme activity (B), and GFP levels (C) as a function of the viral dose (null vector: three rats, 5 x 109 VP: two rats, 8 x 109 VP: three rats, 1 x 1010 VP: seven rats, 5 x 1010 VP: two rats, and 1 x 1011 VP: two rats). Statistical analyses were performed after combining data into three groups (null vector [three rats], low [5 x 109 to 1 x 1010 VP, 12 rats], and high viral dose [more than 1 x 1010 VP, four rats]). *p < 0.05 versus null vector; {dagger}p < 0.05 versus low dose.

 


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  		Figure 6. PET images (A), light microscopic (B), and corresponding fluorescence micrographs (C) obtained in one rat infected with AdCMVnull (left panel) and three rats respectively infected with 1 x 1010, 5 x 1010, and 1 x 1011 VP of AdCMV-mNLS-sr39tk-egfp (panels 2–4 from left to right). The PET images are transverse slices obtained at the mid chest level. Light and fluorescent micrographs (10x magnification) represent identical lung areas in the left lung from adjacent sections. Exposure time for each fluorescent micrograph was the same (960 ms). Fluorescence was mainly seen in distal parenchymal (alveolar) cells, rather than in airway epithelium or microvessel endothelium.

 
Sample PET images obtained from individual rats infected with AdCMVnull and varying doses of AdCMV-mNLS-sr39tk-egfp are presented in Figures 6 and 7 . Similar to the mean values in Figure 5A, there is relatively little difference in signal in the images obtained from the rats administered 1 x 1010 or 5 x 1010 VP but a greater signal in the image from the rats administered the highest dose of viral vector. There was little image slice-to-slice variation (coefficient of variation ± SD, 14.8% + 4.9%), indicating a generally uniform distribution of adenoviral transfection after delivery via surfactant administration. Altogether, by multiplying the known volume of each region of interest with the measured concentration of radiotracer, we calculated that from 0.05 (null vector) to 0.89% of the total injected dose of tracer was taken up by the lungs.



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  		Figure 7. Three-dimensional views of the lungs in a rat infected with 1 x 1011 VP of AdCMV-mNLS-sr39tk-egfp. A movie version of this figure is available in the online supplement.

 
A modest but statistically significant correlation of lung [18F]FHBG uptake (as assessed by PET imaging) was found with both in vitro assays (of mHSV1-TK activity or GFP levels) (Figure 8) . In all cases, lung uptake of [18F]FHBG was greater than that measured in lung regions from rats administered null vector.



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  		Figure 8. Relationships between the lung uptake of [18F]FHBG and mHSV1-TK enzyme activity (A) and GFP levels (B). Each point corresponds to a different rat (n = 19).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates that PET imaging after an intravenous administration of [18F]FHBG is capable of detecting even low levels of mHSV1-tk gene expression in rat lungs. This finding suggests that such methods could be used as a noninvasive reporter system to detect and monitor the effectiveness of therapeutic gene transfer to the lungs, a conclusion supported by the simultaneous and similar correlations between the PET imaging signal on the one hand and the in vitro measures of both mHSV1-TK enzyme activity and GFP levels on the other.

Methodologic Issues
Several PET gene reporter systems have been described, each with their own advantages and drawbacks, as discussed in several recent review articles (17–19). In general, two strategies have been proposed. One is based on the transfer of genes encoding membrane-bound receptors, such as the dopamine-2 receptor (20), not normally expressed in a particular tissue. PET imaging is then used to detect specific binding of a suitable radiolabeled ligand for that receptor.

The other major approach, such as that used in this study, is enzymatic (16, 21). In this case, gene transfer results in the expression of an enzyme that can metabolize a radiolabeled substrate in such a way that the metabolized form is trapped intracellularly. This strategy offers the advantage of signal amplification, as a single copy of the enzyme can metabolize—and trap—many molecules of substrate. However, transmembrane transport of the radiolabeled substrate is also required, which is a factor of potential importance (see below). To date, HSV1-tk is the most extensively studied enzymatic PET reporter system (8, 16, 21–23), with studies showing that successful gene transfer to liver, tumor xenografts, and heart could be detected in mice after intravenous adenoviral vector administration. In addition, recent studies of this system have employed more efficient mutant forms of HSV1-tk, capable of even greater amplification of substrate accumulation (leading to improved PET imaging sensitivity). In this study, we used a newly engineered mHSV1-tk gene (8) to provide the first demonstration that this general strategy for "gene expression imaging" could be used to monitor gene transfer to lung tissue.

Various radiolabeled substrates have also been evaluated as HSV1-tk PET reporter "probes," including acycloguanosine and uracil analogues. Among them, [18F]FHBG and 124I-labeled 2'-fluoro-2'-deoxy-1-ß-D-arabinofuranosyl-5-iodouracil appear to be the best available compounds, combining good sensitivity and specificity (19, 24). In our study, we employed [18F]FHBG as the reporter probe, a substrate with superior trapping properties compared with 124I-labeled 2'-fluoro-2'-deoxy-1-ß-D-arabinofuranosyl-5-iodouracil when sr39tk-based mutants are used as the target PET reporter (8, 9).

Of course, any possibility that PET imaging can be used to measure transgene expression will depend on the accuracy of PET devices to quantify tissue radioactivity. Although an extensive empirical experience substantiates the accuracy of PET in general (25), studies employing the "microPET" generation of scanners are still limited. In this study, Figure 4 shows that a strong linear correlation exists between the radioactivity measurements obtained from imaging data and direct measurements of tissue radioactivity obtained with a {gamma} counter. The regression slope of only 0.36, however, would appear to suggest that the PET measurements systematically underestimated lung radioactivity measurements when compared with direct measurements of radioactivity in lung tissue samples. The absence of correction for photon scattering by the current generation of microPET devices may account for a small underestimation of lung activity measured by PET. However, the apparent underestimation by PET is mainly explained by the differences in the denominator of the two measurements: PET data are expressed per milliliter of tissue volume, whereas the direct tissue radioactivity data are expressed per gram of tissue. Given that in vivo estimates of density in normal lung are typically between 0.3–0.4 g/ml of lung (26), the apparent underestimation is entirely due to this failure to "correct" the PET data differences in tissue density between the in vivo imaging "regions of interest" and the ex vivo tissue samples. Ordinarily, such corrections are easily accomplished by incorporating PET-derived estimates of lung density with "attenuation imaging," an integral component of standard PET imaging protocols (27). However, attenuation images are not yet available with the current generation of microPET devices. Although this type of systematic underestimation affects the slope of the relationship between the PET data and the direct measurements of radioactivity (Figure 4), it does not affect the strength of the correlation with this measurement or, for that matter, with other in vitro measures of gene expression (Figure 8).

In this study, transgene expression was confined virtually entirely to the lung parenchyma as a result of administering viral vector with a surfactant-based method (10, 11). However, given the relatively limited spatial resolution of PET imaging (several mm), it is unlikely that imaging by itself would be able to discriminate expression at different tissue levels within the lungs (e.g., airway epithelium or microvessel endothelium). Accordingly, we anticipate that correlation studies with adjunctive methods, such as GFP fluorescence microscopy as used in this study, will continue to be necessary.

PET Sensitivity to Detect mHSV1-TK Activity in the Lungs
In this study, the PET signal was significantly increased above background (as assessed by imaging after null vector administration) in animals infected by low as well as high doses of adenoviral vector (Figure 5). This was not the case with either in vitro assay of mHSV1-TK enzyme activity or GFP levels, in which gene expression was barely detected at the lower viral doses. This apparent higher sensitivity of PET imaging, in comparison to either in vitro technique, is quite plausible as radionuclide-based methods should be able to detect very low levels of reporter probe accumulation (18), as low as 10-12 mol/L of radiolabeled substrate (28).

Relationship between PET Signal and In Vitro Assays of mHSV1-TK Activity
Optimally, to monitor transgene expression, PET imaging must not only yield accurate measurements of tissue radioactivity, but measurements that are closely related to tissue levels of the gene product. Some studies have reported a highly linear correlation between the PET signal and in vitro assays of transgene expression (21, 23, 29), at least at low levels of mHSV1-tk expression. More recently, however, the possibility that the PET signal may become saturated at high mHSV1-tk expression levels has been raised (16) because cellular entry of the radiolabeled substrates requires potentially saturable nucleosides transporters (23, 24, 30–32). Another factor that may limit uptake of the radiopharmaceutical is the transient nature of the tracer bolus presented to the cells in vivo (e.g., [18F]FHBG is rapidly cleared from the blood). Thus, the pharmacokinetic and transport properties of [18F]FHBG in vivo may limit the PET signal when compared with in vitro assays of mHSV1-TK enzyme activity in which substrate delivery is not limited. Our data are consistent with these possibilities in that we measured relatively little additional PET signal as mHSV1-tk gene expression increased (as assessed by in vitro methods) (Figure 5A).

It is also possible that differences in how the substrate is handled by different organs could affect overall uptake. [18F]FHBG is partly excreted by the liver; thus, its concentration may remain relatively high in this organ during the time of excretion, increasing the contact time between tracer and cells expressing mHSV1-tk. This difference might account for differences in the correlation with in vitro activity between studies where liver has been the target organ (16, 23, 29) versus the lungs as in this study. Clearly, these issues of substrate type, availability and transport will require additional study.

Clinical Implications
In this study, we described a noninvasive imaging strategy that can be used to detect, quantify, and describe the spatial distribution of transgene expression within the lungs. Thus, with systems such as the one employed in this study, it should be possible to monitor the onset and duration of transgene therapies noninvasively over time. Recent advances in transgene vectors as well as improvements in promoter technology have expanded the potential application of gene transfer therapy from inherited monogenic to acute diseases (1). Thus, a wide variety of lung diseases are potentially treatable by gene therapy, including pulmonary hypertension (33), cystic fibrosis, and the acute respiratory distress syndrome (2, 10), among others. The PET reporter system described in this study should be viewed as a "first-generation" system, one that will undoubtedly improve as alternative reporter genes, probes, and PET instruments are developed and evaluated. Nevertheless, the potential value of such a system in both experimental and clinical studies is obvious. Even current generation "microPET" systems (as used herein) are able to detect and quantify transgene expression in murine models, thereby permitting studies in already available genetic models of disease. In addition, the possibility that this imaging strategy can be deployed clinically has been advanced by a recent report documenting the safety of [18F]FHBG administration to healthy volunteers (34). Ultimately, however, it will not only be important to demonstrate robust correlations between reporter gene and therapeutic gene expression, but also to document that such expression—as evaluated by imaging—has a predictable relationship to the intended physiologic effect of the therapeutic transgene.

Conclusion
PET imaging is a sensitive and quantitative method for detecting pulmonary reporter gene expression noninvasively.


   Acknowledgments
 
The authors thank Jim Kozlowski and Matt Bernstein for their contribution to this study and gratefully acknowledge the support provided by the microPET facility staff in the Division of Radiological Sciences.


   FOOTNOTES
 
Supported by National Institutes of Health HL32815, HL13851, P50CA94056, and R24 CA83060.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form October 24, 2002; accepted in final form December 26, 2002


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 METHODS
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