Repetitive Imaging of Reporter Gene Expression in the Lung

Abstract

Positron emission tomographic imaging is emerging as a powerful technology to monitor reporter transgene expression in the lungs and other organs. However, little information is available about its usefulness for studying gene expression over time. Therefore, we infected 20 rats with a replication-deficient adenovirus containing a fusion gene encoding for a mutant Herpes simplex virus type-1 thymidine kinase and an enhanced green fluorescent protein. Five additional rats were infected with a control virus. Pulmonary gene transfer was performed via intratracheal administration of vector using a surfactant-based method. Imaging was performed 4–6 hr, and 4, 7, and 10 days after gene transfer, using 9-(4-[¹⁸F]-fluoro-3-hydroxymethylbutyl)guanine, an imaging substrate for the mutant kinase. Lung tracer uptake assessed with imaging was moderately but significantly increased 4–6 hr after gene transfer, was maximal after 4 days, and was no longer detectable by 10 days. The temporal pattern of transgene expression measured ex vivo with in vitro assays of thymidine kinase activity and green fluorescent protein was similar to imaging. In conclusion, positron emission tomography is a reliable new tool to evaluate the onset and duration of reporter gene expression noninvasively in the lungs of intact animals.

Keywords

Positron emission tomography FHBG reporter gene herpes simplex virus 1 thymidine kinase green fluorescent protein

Introduction

Pulmonary gene therapy is still being actively pursued as a treatment for both inherited disease and acute rapidly progressive diseases [1,2]. The continuing interest in this therapeutic strategy stems from the potential to selectively modify gene expression, and therefore, cell physiology, in target organs such as the lungs.

Eventually, as gene therapies become ready for translation into the clinical setting, it will be important to have noninvasive methods available to evaluate transgene expression in vivo. In particular, the ability to monitor transgene expression repetitively over time will be needed to detect the onset and duration of gene expression as well as the relationship of the gene product to its putative physiologic effects. For instance, if gene therapy is to be used for acute but self-limited disease, high levels of the “therapeutic” protein would ideally be obtained shortly after vector delivery. Furthermore, the duration of expression would be transient so as to leave cell physiology unchanged after complete recovery from the acute disease. The goal of imaging would be to document such events accurately and noninvasively.

A number of imaging technologies are currently under investigation as potential new tools for monitoring reporter gene expression noninvasively. Among these are radionuclide-based methods, such as positron emission tomography (PET). These techniques have a number of attractive features, including high sensitivity, absolute signal quantitation, and the potential for direct translation into the clinical setting. Several PET reporter systems [3–6] have been described; we have recently reported the use of one such system in rat lungs, using a mutant version of the Herpes simplex virus type-1 thymidine kinase (mHSV1-tk) as a reporter gene and 9-(4-[¹⁸F]-fluoro-3-hydroxymethylbutyl)guanine ([¹⁸F]FHBG) as an imaging substrate [7].

To date, only a few experimental studies have used any of these new imaging technologies to determine the chronology of transgene expression in vivo after adenoviral-mediated gene transfer [8–12]. However, none of these studies has specifically targeted the lungs. More importantly, none of the time-series data in these studies has been validated against tissue-based assays of reporter gene expression. It is possible, for instance, that the accuracy of the imaging method could degrade as individual animals are repeatedly studied. Thus, the current study was designed to determine the utility of PET imaging to study the kinetics of adenovirus-mediated gene transfer into the lungs.

Material and Methods

Replication-deficient (Ela/E3 deleted) recombinant human type 5 adenoviruses, containing a fusion gene encoding for a mutant Herpes simplex virus type-1 thymidine kinase [6] and an enhanced green fluorescent protein (Ad-CMV-mNLS-HSV1sr39tk-egfp), were constructed as described elsewhere [7]. A control vector (AdCMVnull) was constructed with a shuttle vector containing no cDNA.

Experimental Protocol

The present study was approved by the Washington University School of Medicine's Animal Studies Committee and was performed on 25 Sprague-Dawley rats (mean weight ± SD = 277 ± 26 g), assigned to two experimental groups: a control group (n = 5) infected with 5 × 10¹⁰ viral particles (VP) of AdCMVnull and another group (n = 20) infected with 5 × 10¹⁰ VP of Ad-CMV-mNLS-HSV1sr39tk-egfp. Adenovirus delivery to rat lungs was performed using a surfactant-based method as previously described [7,13]. Nine animals (5 in the AdCMVnull group and 4 in the Ad-CMV-mNIS-HSV1sr39tk-egfp group) were repeatedly studied with PET 4–6 hr, and 4, 7, and 10 days after gene transfer (Figure 1). Four additional rats were euthanized at each of these time points after PET imaging for in vitro assessment of pulmonary transgene expression (see below).

Figure 1.

Experimental design. The reference date (Day 0) corresponds to the time of lung gene transfer. Ad-CMV-mNLS-HSV1sr39tk-egfp = replication-deficient adenovirus encoding for a mutant herpes simplex virus 1 thymidine kinase and an enhanced green fluorescent protein; AdCMVnull = control virus.

PET Studies

[¹⁸F]FHBG was synthesized using a microwave-mediated method generating high chemical yield and purity [14], with specific activity ranging between 2000 to 3000 Ci/mmol. PET imaging was performed on a microPET-R4 camera [15]. Rats were anesthetized with isoflurane shortly before intravenous injection of 1 ± 0.2 μCi/g of b.w. (37 ± 6 kBq/g) of [¹⁸F]FHBG. After one hour to allow for tracer clearance from blood, each animal was placed in the supine position into the micro-PET scanner, with image acquisition over a period of 15 min.

Regions of interest (ROIs) were drawn on the left and right lungs using Analyze v4.0 for image analysis [16]. All radioactivity 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 ROI were normalized to the injected dose (ID) of [¹⁸F]FHBG.

In Vitro Assays

Immediately after PET scanning was completed, rats were deeply anesthetized by an overdose of ketamine and xylazine. After thoracotomy, the lung circulation was perfused with 0.9% saline via the spontaneously beating right ventricle. The lungs were then harvested and frozen in liquid nitrogen.

Thymidine kinase enzyme activity in lung tissue extracts was assessed on 1 μg of protein, as previously described [7,17]. Briefly, 8-[³H] penciclovir was added to the preparation and its phosphorylated compound was quantified in an anion exchanger filter using a beta counter. Results were then expressed as a percentage of 8-[³H] penciclovir phosphorylation/μg protein/min after normalization to the total amount of substrate and to the enzyme–substrate exposure time. GFP levels in lung tissue extracts were quantified with ELISA as previously described [7].

Statistical Analysis

PET tracer uptake was compared between groups and time points by two-way repeated-measures ANOVA. Comparison of in vitro assay levels between time points was performed by ANOVA on ranks. For additional pairwise comparisons, Tukey's or Dunn's test was performed where appropriate. The level of significance was set at p ≤ .05. Statistical analysis was performed using SigmaStat (SPSS, Chicago, IL, USA).

Results

Sequential PET images of two animals, which were representative of their experimental groups, are presented in Figure 2. Although the intrathoracic radioactivity was not different from background for the control rat (upper panel), lung boundaries were easily detectable on PET images obtained 4–6 hr, 4 days, and 7 days after gene transfer (bottom). However, 10 days after gene transfer, no radioactivity in excess of background was detectable in the lungs of either animal.

Figure 2.

Repetitive pulmonary imaging with micro-PET of reporter gene expression in two animals representative of their experimental groups [one rat infected with Ad-CMV-mNLS-HSV1sr39tk-egfp and an animal infected with the control virus (AdCMVnull)]. Images are transverse sections at the mid-chest level and ROIs were drawn to represent lung boundaries. Ad-CMV-mNLS-HSV1sr39tk-egfp = replication-deficient adenovirus encoding for a mutant herpes simplex virus 1 thymidine kinase and an enhanced green fluorescent protein.

Overall, quantitative information obtained from PET images for all the animals confirmed the visual impressions of Figure 2. There were no significant slice-to-slice, or ventral–dorsal regional differences in tracer uptake over time (data not shown). After averaging values from all ROIs in each individual rat, and then averaging values from all rats at any one time, lung tracer uptake assessed with PET was moderately but significantly increased as soon as 4–6 hr after gene transfer, was maximal after 4 days, and was only slightly different from the control group 7 days after viral infection with the adenovectors (Figure 3). Ten days after vector administration, no mHSV1-TK enzyme activity was detectable with PET imaging in the lungs. Mean values at Day 4 in animals studied repetitively were significantly different (p < .05) from mean values at Days 0, 7, and 10. Furthermore, the results in the group of animals studied repetitively replicated the results in individual groups studied once prior to tissue harvesting for in vitro confirmation of reporter gene activity (Figure 3).

Figure 3.

Evolution of the lung PET signal over time. Bars are standard deviation. For clarity, only one side of the two-sided standard deviation is shown. ^*p < .05 versus AdCMVnull group, ^†p ≤ = 05 versus Day 10. [¹⁸F]FHBG = 9-(4-[¹⁸F]-fluoro-3-hydroxymethylbutyl)guanine; ID = injected dose; Ad-CMV-mNLS-HSV1sr39tk-egfp = replication-deficient adenovirus encoding for a mutant herpes simplex virus 1 thymidine kinase and an enhanced green fluorescent protein; AdCMVnull = control virus.

Figures 4 and 5 show that the temporal pattern of transgene expression measured with PET imaging was similar to that measured with in vitro assays of mHSV1-TK enzyme activity and GFP levels. Four to six hours after gene transfer, a statistically significant increase in thymidine kinase enzymatic activity was detected in vitro in the lungs, while there was only a trend toward an increase in GFP levels at this time point. By all three methods (imaging, mHSV1-TK activity assays, and GFP levels), reporter gene expression peaked 4 days after gene transfer, was slightly but consistently increased in all but one animal on Day 7, and was not detectable 10 days after gene transfer. The absence of significant reporter gene levels on Day 7 is probably due to the number of animals studied, since all methods (imaging, mHSV1-TK activity assays, and GFP ELISA) detected above-background levels of transgene expression in all but one animal. Accordingly, when the results of both imaging groups were combined (n = 8, Figure 3), the mean value (0.044 ± 0.014 %ID/mL lung) was statistically different (p < .05) than background activity measured in the control group that only received the null vector (0.025 ± 0.004 %ID/mL lung).

Figure 4.

Evolution over time of pulmonary reporter gene expression assessed by in vitro assay of mHSV1-TK enzyme activity. Bars are standard deviation. ^*p < .05 versus AdCMVnull group, ^†p < .05 versus Day 10. Ad-CMV-mNLS-HSV1sr39tk-egfp = replication-deficient adenovirus encoding for a mutant herpes simplex virus 1 thymidine kinase and an enhanced green fluorescent protein; AdCMVnull = control virus; PCV = penciclovir.

Figure 5.

Evolution over time of pulmonary reporter gene expression assessed by green fluorescent protein levels (ELISA). Bars are standard deviation. ^*p < .05 versus AdCMVnull group, ^†p ≤ .05 versus Day 10. Ad-CMV-mNLS-HSV1sr39tk-egfp = replication-deficient adenovirus encoding for a mutant herpes simplex virus 1 thymidine kinase and an enhanced green fluorescent protein; AdCMVnull = control virus.

Discussion

Methodologic Issues

The concept of using PET imaging to follow transgene expression is based on combinations of so-called imaging reporter genes and imaging reporter probes [18]. The PET reporter–probe system used in the present study is a variant of the one originally described by Tjuvajev et al [19] and was later developed by this same as well as other groups [20]. Subsequently, this system was progressively refined by genetic engineering of several different mutant thymidine kinases [4,6] and by developing new probes with increased enzymatic affinity for these proteins, such as [¹⁸F]FHBG [21]. These modifications have resulted in an improved sensitivity for this particular reporter–probe system [12], allowing gene expression to be quantified in several different target tissues [12,22,23].

Even though various factors would be expected to limit the quantitative accuracy of gene expression in the lungs with PET imaging (e.g., partial-volume effect, lung motion, tissue density heterogeneity), we nevertheless found in a previous study that the PET signal correlates with in vitro assays of reporter gene expression in lung tissue samples [7]. The present study supports these earlier observations since the lung imaging signal and in vitro assays of transgene expression share similar patterns over time (Figures 3–5).

It was also important in the present study to demonstrate that repetitive PET measurements in the same animal remained accurate, for example, to rule out a potential influence of repetitive administration of the radioactive probe on lung tracer uptake. Given the relatively short half-life of ¹⁸F (110 min), insufficient radioactive decay between time points would not explain persistent radioactivity on successive PET images as a minimum of 3 days was allowed between PET data acquisitions. On the other hand, depending on the mass of substrate administered, repetitive administration of [¹⁸F]FHBG could theoretically saturate nucleoside transporters preventing tracer entry into target cells. However, given the high specific activity of tracer synthesis used in this study, we estimate the mass of [¹⁸F]FHBG to be only ˜0.2–0.8 pmol/g of b.w. The fact that the lung PET signal was not statistically different at any one time between the group of animals repeatedly studied and the groups that were studied only once (Figure 3) also supports the continued accuracy of the method over time.

Chronology of Adenovirus-mediated Transgene Expression in the Lungs

The possibility of using noninvasive imaging to study the onset and duration of transgene expression is obviously an attractive one, for both gene therapeutics and for studies which seek to determine relationships between the effectiveness of gene transfer and the biological effects of the gene product. The present study is the first use of PET imaging to determine the onset of transgene expression after adenovirus-mediated gene transfer. Significant levels of the transgene were detected in the lungs as soon as 4–6 hr after the gene transfer procedure. The imaging results were validated with two in vitro assays of transgene expression. Although contamination of the viral preparation with either reporter protein or mRNA could theoretically explain such early detection of the transgene (so-called pseudotransduction), this phenomenon has so far only been observed with retroviruses and adeno-associated virus and never with cesium chloride-purified adenoviruses [24]. Furthermore, using the same methods to generate virus as were used in the present study, Dumasius et al. [24] have also reported physiologically significant levels of human β₂-adrenergic receptor in the lungs within hours after adenovirus-mediated gene transfer without evidence for pseudotransduction. Together, these results confirm the high efficiency of adenoviral vectors and suggest that they may be particularly appropriate for therapeutic gene transfer during acute disease. They also demonstrate the usefulness of this imaging strategy to determine the onset of transgene expression.

Only a few studies, however, have used PET imaging to study the “magnitude and duration” of adenoviral-mediated gene transfer. In our study, peak levels of gene expression were obtained on Day 4 (0.24 ± 0.07 %ID/mL lung, combining data from both sets of rats in Figure 3 at this time point). The magnitude of expression, as measured by PET, was similar to that observed in a previous study (0.19 ± 0.00 %ID/mL lung) [7] 3 days after vector administration in animals given a similar dose of adenovirus as in the current study. However, an issue of concern is that the PET imaging signal at Day 4 only increased by a factor of 2–3 over Day 0 measurements (Figure 3), while the in vitro assays (Figures 4 and 5) showed increases of 50–150-fold. This observation raises the possibility that the PET imaging signal may not fully track the “magnitude” of gene expression, especially in tissues with high levels of expression—an issue which will need to be addressed more fully in future studies.

Similar to these findings (Figures 3–5), Inubushi et al. [23], using first-generation adenoviral vectors and an expression cassette driven by a CMV promoter, found that myocardial reporter gene expression fell off dramatically 7–10 days after gene transfer in rats. In two other studies, PET imaging was used to detect transgene expression in the liver of a small number of mice for as long as 90 days after adenovector administration [11,12]. It should be noted that none of these studies by others validated their results against tissue-based assays. Overall, however, the reasons for the differences in the duration of transgene expression among these different studies may relate to differences in species used, tissue transduced, or the inflammatory response invoked by the adenovector (see below). Regardless, such differences again demonstrate the utility of imaging to study the factors underlying them.

Instead of imaging, a number of studies have used tissue-based assays to specifically monitor the duration of pulmonary transgene expression. In general, after intratracheal vector administration, a peak of transgene expression is observed 1 to 3 days after first-generation adenovirus infection, with little detectable expression in the lungs after 10–14 days [13,25–29] (although occasional studies have reported pulmonary transgene expression for 14 or more days after adenovector administration [30,31]. Our results are similar to the majority of these reports.

Relatively transient transgene expression after adenovirus-mediated gene transfer has been linked to a cytotoxic T-cell response to MHC-1 adenoviral antigens, which leads to rapid clearance of transduced cells. Such a response is the most likely explanation for the relatively short duration of gene expression in the current study, and indeed, more prolonged transgene expression in target tissue is possible in immune-deficient mice [12,25,27] or in immune-competent animals after infection with newer generation adenovectors that are deleted of all adenoviral genes (“gutless” or helper dependent) [32–34]. The current results indicate that imaging could be used to follow the duration of gene expression with these newer vector technologies.

Additional factors may also contribute to an apparent variation in the duration of transgene expression after gene transfer mediated with first-generation adenovectors, including the specific target tissue [9,35], the species (mice [9,35] vs. rats), or differences in the immunogenicity of transgenic proteins. The reporter gene promoter may be another source of variation—the Rous sarcoma virus promoter, for example, being associated with more prolonged transgene expression in target tissue than the CMV promoter [28,36]. Differences between half-lives of reporter mRNA or proteins may also affect the kinetics of transgene expression at the protein level [37]. Finally, the sensitivity of the biological method quantifying transgene expression must be considered. Indeed, low-sensitivity methods will underestimate the apparent duration of reporter gene expression while high-sensitivity methods may detect physiologically insignificant levels of the transgene. We have previously demonstrated that the PET reporter system used in this study is at least as sensitive as in vitro assessments of thymidine kinase enzymatic activity or GFP quantitation by ELISA [7], but the biological significance of the detection threshold of these three techniques is unknown. PET imaging, however, has the advantage of detecting only functional reporter proteins, while other methods (e.g., Northern blots, Western blots, ELISA) may detect nonfunctional copies of transcript or proteins. Thus, a variety of factors, both technical and biological, can affect the evaluation of transgene expression kinetics.

In addition to PET imaging, optical imaging is also emerging as one of a number of potentially attractive, highly sensitive, and low-cost approaches for real-time analyses of reporter genes. Using this method, detectable levels of transgene were still found either 14 days (myocardium [10]), 30 days (liver, salivary glands [8]), or 150 days (skeletal muscles [9]) after adenoviral-mediated gene transfer. Once again, this apparently prolonged duration may be the result of species differences, organ differences, or differences in method sensitivity (PET vs. optical imaging), among others.

Experimental and Clinical Implications

One immediate consequence of the results of the present study is that experimental studies that seek to determine the onset and duration of transgene expression can be conducted much more efficiently with a PET imaging strategy than methods that depend on tissue retrieval. The ability to perform serial measurements should also help the evaluation of gene delivery systems. Perhaps even more importantly, these results provide additional support for a direct translation of this imaging strategy into the clinical setting. In this connection, several PET reporter probes have already been tested in humans, with promising results in terms of biosafety, stability, and radiation dosimetry [38,39]. To date, however, a direct correlation between PET imaging of a reporter gene and the physiologic effect of a linked “therapeutic gene” is still lacking. Such information is still required to fully demonstrate the potential of PET imaging for monitoring in gene therapy protocols.

Conclusion

PET imaging provides useful information on reporter gene expression over time in the lungs. These results add to a growing body of evidence that this imaging strategy is a promising approach for eventually monitoring pulmonary gene therapy in clinical trials.

Footnotes

Acknowledgments

We thank Jim Kozlowski and Kathryn Akers for their contribution to this study and gratefully acknowledge the support provided by the micro-PET facility staff in the Division of Radiological Sciences of Washington University School of Medicine. Funding sources: NIH HL32815, HL 66211.

Abbreviations:

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