Non-invasive radioiodine imaging for accurate quantitation of NIS reporter gene expression in...

1NON-INVASIVE RADIOIODINE IMAGING FOR ACCURATEQUANTITATION OF NIS REPORTER GENE EXPRESSION INTRANSPLANTED HEARTS

Davide Ricci*,***, Ari A Mennander*,****, Linh D Pham**, Vinay P Rao*, Naoto Miyagi*, GuerardW Byrne*, Stephen J Russell**, and Christopher GA McGregor*

*Mayo Clinic William J von Liebig Transplant Center, Rochester, Minnesota 55905 USA **MolecularMedicine Program, Mayo Clinic College of Medicine, Rochester, Minnesota 55905 USA ***Division ofCardiac Surgery, Foundation I.R.C.C.S Policlinico San Matteo, University of Pavia, Pavia, Italy ****HeartCenter, Tampere University Hospital, Finland

AbstractObjectives—We studied the concordance of transgene expression in the transplanted heart usingbicistronic adenoviral vector coding for a transgene of interest (human carcinoembryonic antigen:hCEA - beta human chorionic gonadotropin: βhCG) and for a marker imaging transgene (humansodium iodide symporter: hNIS).

Methods—Inbred Lewis rats were used for syngeneic heterotopic cardiac transplantation. Donorrat hearts were perfused ex vivo for 30 minutes prior to transplantation with University of Wisconsin(UW) solution (n=3), with 109 pfu/ml of adenovirus expressing hNIS (Ad-NIS; n=6), hNIS-hCEA(Ad-NIS-CEA; n=6) and hNIS-βhCG (Ad-NIS-CG; n=6). On post-operative day (POD) 5, 10, 15all animals underwent micro-SPECT/CT imaging of the donor hearts after tail vein injection of 1000μCi 123I and blood sample collection for hCEA and βhCG quantification.

Results—Significantly higher image intensity was noted in the hearts perfused with Ad-NIS (1.1±0.2; 0.9±0.07), Ad-NIS-CEA (1.2±0.3; 0.9±0.1) and Ad-NIS-CG (1.1±0.1; 0.9±0.1) compared toUW group (0.44±0.03; 0.47±0.06) on POD 5 and 10 (p<0.05). Serum levels of hCEA and βhCGincreased in animals showing high cardiac 123I uptake, but not in those with lower uptake. Abovethis threshold, image intensities correlated well with serum levels of hCEA and βhCG (R2=0.99 andR2=0.96 respectively).

Conclusions—These data demonstrate that hNIS is an excellent reporter gene for the transplantedheart. The expression level of hNIS can be accurately and non-invasively monitored by serialradioisotopic single photon emission computed tomography (SPECT) imaging. High concordancehas been demonstrated between imaging and soluble marker peptides at the maximum transgeneexpression on POD 5.

*Address for correspondence: Christopher G. A. McGregor, MB, FRCS, Director, Mayo Clinic William J von Liebig Transplant Center,9-233 Charlton Building, Rochester, MN 55905 USA, Telephone: (507) 255-6038, Fax: (507) 255-4500, e-mail:[email protected] results of this work will be presented as an oral presentation at the 21st Annual Meeting of the European Association of Cardio-Thoracic Surgery, 15-19 September 2007, in Geneva.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptEur J Cardiothorac Surg. Author manuscript; available in PMC 2008 April 1.

Published in final edited form as:Eur J Cardiothorac Surg. 2008 January ; 33(1): 32–39.

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KeywordsGene therapy; heart transplantation; sodium iodide symporter (NIS); single photon emissioncomputed tomography (SPECT); molecular imaging

IntroductionAdvances in nuclear imaging techniques have made molecular imaging of gene transductionfeasible with the use of reporter genes like herpes simplex virus thymidine kinase (HSVtk),human norepinephrine transporter (hNET) and human sodium/iodide symporter (hNIS).Ideally, the reporter gene is expressed on human cells but not on the organ being targeted andis therefore non-immunogenic.

Sodium iodide symporter (NIS) is an integral plasma membrane glycoprotein that mediatesactive iodide transport into the thyroid follicular cells, the first step in thyroid hormonebiosynthesis. NIS-mediated thyroidal iodide transport from the bloodstream to the colloid is avectorial process made possible by the selective targeting of NIS to the basolateral membrane.NIS also mediates active iodide transport in other tissues, including salivary glands, gastricmucosa, and lactating mammary glands, in which it translocates iodide into the milk for thyroidhormone biosynthesis by the nursing newborn. NIS provides the basis for the effectivediagnostic and therapeutic management of thyroid cancer and its metastases with radioiodide.

Now that NIS has been cloned and characterized, it may be possible to develop novel strategiesto differentially modulate NIS expression and/or activity, enhancing it in target tissues andimpeding it in others [1,2].

A vital step in transgenic animal studies and gene therapy clinical trials is the ability to monitorthe extent of transgene expression. Therefore, it is pertinent to develop techniques to noninvasively and repetitively determine the location, duration, and magnitude of transgeneexpression in living animals. Several studies and clinical trials have already successfullydemonstrated the possibility to use therapeutic gene encoded into viral vectors but in none hasbeen possible to monitor in vivo gene expression [3–5]. In any gene therapy study we shouldknow the number, distribution and fate of the genetically modified cells, and the kinetics ofproduction of the therapeutic protein over time.

It is possible to use NIS as an imaging reporter gene to monitor the expression profile of thetransgene in transgenic animal models. The effectiveness of NIS as a non-invasive reportergene has already been validated by several studies in tumor models of gene transfer [6,7].

The expression of NIS on mammalian thyroid follicular cells makes it an attractive reportergene due to the potentially attenuated immune response to the gene product. In addition theabsence of NIS on the cardiomyocytes makes it an interesting myocardial reporter gene. Thetransduction of NIS encoded into an adenovirus did not determine myocardial injury ordysfunction in previous in vitro studies [8].

In this study we sought to establish the validity of a novel approach to quantitative monitoringtransgene expression in the transplanted heart. The approach is to use a vector coding both forthe transgene of interest and for a marker transgene whose expression level can be determinedby radioisotopic imaging studies. If there is concordance between the expression levels of thetwo transgenes, the expression level of one can be inferred from the measured expression levelof the other. The confirmation of this concordance would lead to future applications of cardiactherapeutic genes with the possibility to monitor over time gene expression. In this way wecould easily identify the reasons for treatment failure, whether due to inadequate transduction

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of target cells/inadequate virus dose (little or no gene expression), inadequate virus replication(persistent low level or falling gene expression) or myocardial cell resistance to virus mediatedeffect despite adequate virus spread (continuously rising expression).

We used bicistronic adenoviral vectors coding for hNIS and either the soluble extracellulardomain of human carcinoembryonic antigen (hCEA) or the beta chain of human chorionicgonadotropin (βhCG). Non-invasive imaging of radioiodine uptake and serum quantificationof soluble markers were performed with the use of computed tomography (CT) co-registeredwith micro-SPECT [9] following adenoviral gene transfer in a rat model of cardiactransplantation using three adenoviral vectors: Ad-hNIS, Ad-hNIS-hCEA and Ad-hNIS-βhCG.

Materials and MethodsInbred male Lewis rats (250–350g, Harlan®, IN) were used as donors and recipients forsyngeneic heterotopic heart transplants. Procedures and handling of animals were reviewedand approved by the Institutional Animal Care and Use Committee of the Mayo Clinic andFoundation in compliance with “Principles of Laboratory Animal Care” formulated by theNational Society for Medical Research and the “Guide for the Care and Use of LaboratoryAnimals” prepared by the Institute of Laboratory Animal Resources and published by theNational Institutes of Health (National Institutes of Health publication No. 86–23, revised1996).

Recombinant adenovirus productionHuman CEA, βhCG and hNIS were ligated into human cytomegalovirus (CMV) shuttle vector.Once in the CMV vector, the CMV promoter hCEA/hCG gene was ligated into an E1/E3deleted adenoviral serotype 5 (Ad) vector at E3 position within Csp451 site. Then CMVpromoter-hNIS was ligated into Ad vector at E1 position after digested both with I-CeuI andPI-SceI to make Ad-hCEA3-NIS1 and Ad-hCG3-NIS1. All viral vectors were purified usingstandard methods of ethanol precipitation, phenol-chloroform extraction, mini-prep, and gelextraction. All steps were screened using gel electrophoresis to ensure all reactions were carriedout properly and had the desired results.

Once successful cloning and purification of the Ad vector had been completed, the vector waslinearized using Pac-1 and transfected into 293 human embryonic cells. The 293 cells wereinfected using a modified protocol from the Polyfect transfection kit. After well developedplaques were formed, the cells were harvested and went through three rounds of freeze thawingbefore being applied to 293 cells again. This step was done three times in order to amplify thevirus. Once this was completed, the virus was rescued using cesium chloride extractionmethods. This followed by using dialysis against 10% glycerol in phosphate buffer saline (PBS)twice in order to collect the purified virus.

Purified virus was diluted in cold University of Wisconsin (UW) solution at a concentrationof 109 pfu/ml just prior to ex vivo perfusion of donor hearts. Viral titers were determined byplaque assay and expressed as plaque forming units per ml (pfu/ml).

Donor operationThe donor hearts were harvested as previously described [10]. Briefly, after anesthesia(pentobarbital sodium 70 mg/kg administered intraperitoneally), a bilateral thoracotomy wasperformed and 200 U of aqueous heparin injected into the inferior vena cava. The innominateartery was cannulated with a 24-gauge cannula, and the venae cavae and pulmonary veins wereligated en bloc with 6-0 silk. The aorta was clamped distal to the cannula, and the heart was

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arrested with an infusion of cold UW into the aortic root through the cannula placed into theinnominate artery. After harvesting, the heart was stored in the same cardioplegic solution at4°C.

Experimental groups and gene transferTwenty-one transplants were performed within four groups. In the first group (n=3), the donorhearts harvested as previously described [10] were perfused for 30 minutes at 4°C with 5 mlof UW solution using the Langendorff perfusion apparatus. In the second group (n=6), donorhearts were perfused with 5 ml of Ad-NIS diluted in UW solution at a concentration of 1×10 9 pfu/ml under the same conditions. In groups 3 and 4 (n=6 in each group) donor heartswere perfused respectively with Ad-NIS-CEA and Ad-NIS-CG following the same protocol.Hearts from all groups were then stored in UW solution at 4°C while the recipient was prepared.All the animals underwent micro-SPECT/CT imaging of the donor hearts after tail veininjection of 1000 μCi 123I on post-operative day (POD) 5, 10 and 15 following gene transfer.On the same post-operative days 400 μl of blood sample were obtained for hCEA and βhCGserum quantification. A control group using only the adenoviral vector with no gene encodedwas not included in this study because in a previous study from our group (Rao VP, et al inpress) no differences were found in iodine uptake between hearts perfused with solutioncontaining the adenovirus + no gene (Ad-Null) and only UW perfused hearts.

Recipient operationFollowing anesthesia (pentobarbital sodium 70 mg/kg administered intraperitoneally) amidline laparotomy was performed and the abdominal aorta and the inferior vena cava exposedbelow the origin of the renal vessels. The donor heart was transplanted into the recipient byend-to-side anastomoses of the aorta and the pulmonary artery to the abdominal aorta andinferior vena cava, respectively, using 8-0 monofilament sutures. During surgery, the heart waswrapped in gauze and kept cold by use of topical ice-cold saline solution. All rats receivedanalgesia postoperatively (Buprenorphine administered subcutaneously) and recovered withoxygen in a warm environment. Function of the graft was checked daily by palpation of thebeating transplanted heart.

Imaging with micro-SPECTAll animals were imaged on POD 5, 10 and 15, under sedation (intraperitoneal pentobarbital50mg/kg). 37 MBq of 123I was injected into the tail vein and micro-SPECT/CT imagingcommenced after 20 minutes. Syringe activity was measured prior to and post-injection toquantify the injected dose accurately. Micro-SPECT images were obtained under a high-resolution gamma camera (X-SPECT, Gamma Medica-Ideas Inc., CA) with a low energy highresolution parallel hole collimator with 64 projections at a rate of 10s per projection and anacquisition time of 13m 46s. CT images were obtained under a circular orbit at a thickness of50μM per slice under the same scanner without moving the animal in between. This ensuredaccurate co-registration of the axial CT images with the micro-SPECT maps.

In Vivo image analysisSPECT and CT images were stored and pixel-intensity of the images was quantified asdescribed in earlier studies [9]. Briefly, the regions of interest (ROI) were defined manuallyaround the heart shadows on the reconstructed axial tomographic images in all sections inwhich the heart was visualized, and quantification performed on the co-registered micro-SPECT images. Cumulative pixel counts, within the defined ROI, were converted to activityusing a conversion factor of 1.63 × 10−7 MBq/measured count derived from our previousstudies. Values were expressed as a percentage of the injected dose per gram of heart tissue(%ID/g).

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Measurements were obtained by an independent observer blinded to the origin of the images.The threshold value for image intensity (%ID/g) was estimated from the UW only perfusedhearts data pooled across the three time points (POD 5, 10 and 15), which represented the rangeof baseline amount. Since this cut-off served to differentiate baseline versus virus-induced %ID/g, the maximum value from the baseline data plus one standard deviation was chosen asthe threshold value. Serum quantification of soluble markers hCEA and βhCG was performedon blood collected from all the recipients on POD 5, 10 and 15 before each 123I injection andmicro-SPECT/CT imaging. Following the last imaging procedure, the rats were euthanized byexsanguination and the transplanted hearts explanted and weighed.

Anti-hCEA and anti-βhCG antibody screeningAnti-hCEA and anti-βhCG antibodies were analyzed by ELISA. Wells were coated overnightat 4°C with 200 ng/ml hCEA or βhCG (Scripps Laboratory, San Diego, California). The plateswere washed and serial dilutions of rat serum were added. Bound antibodies were detectedusing horseradish peroxidise-conjugated antibodies and substrate [11]. The test was performedon all the serum samples collected at POD 5, 10 and 15.

StatisticsWe assessed the correlation of %ID/g with hCEA and βhCG both at each time point and overallusing univariate linear regression. Specifically, we modeled %ID/g using hCEA as thepredictor among Ad-NIS-CEA rats and, separately, βhCG as the predictor among Ad-NIS-CGrats. The model p-value and R2 value were reported. The sensitivity of each parameter, stratifiedby day, was computed using known cut-off values for hCEA and βhCG serum levels, and anestimated cut-off value for %ID/g. The cut-off value for presence of viral %ID/g was estimatedfrom the UW group (control) data pooled across the three time points, which represented thebaseline range. Since the cutoff value would serve to differentiate baseline versus virus-induced%ID/g, the maximum value from the baseline data plus one standard deviation was chosen asthe estimate. For those in the three Ad-NIS groups, sensitivity of %ID/g was computed as thepercentage of rats over the threshold of 0.6. For hCEA, sensitivity was calculated as thepercentage of rats in the Ad-NIS-CEA group above the cut-off value of 0.1 ng/ml. Likewise,sensitivity of βhCG was computed as the percentage of rats over the cutoff of 0.5 IU/L in theAd-NIS-CG group. Each sensitivity estimate per parameter above was calculated at the threemeasurement days and over all three days.

We further evaluated each of the three parameters’ ability to distinguish rats that were perfusedwith the virus versus those that were not at various days using logistic regression. To predictthose with a specific virus, we used the corresponding parameter measurement on each andthen over all three days. The c-statistic from the logistic regression, a measure gauging themodel’s discriminatory ability, was reported for each parameter at the study time points. Inaddition, standard errors for the c-statistic were calculated using the DeLong method allowingformal comparisons between pairs of three parameter c-statistics. P-values were obtainedassuming the three c-statistic estimates were normally distributed, and thus that the differencebetween any two could also be considered normal. A significance level of 0.05 was used forall analysis.

ResultsSPECT/CT image intensity quantification

All animals underwent SPECT/CT sequential imaging after tail vein injection of 1000μCi 123I on POD 5, 10 and 15. Recipients were sacrificed following the imaging protocol onday 15, transplanted heart weight was measured and intensity of images obtained of eachtransplanted heart was quantified as described in Material and Methods.

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Significantly higher image intensity was noted in the hearts perfused with Ad-NIS (1.1±0.2;0.9±0.07), Ad-NIS-CEA (1.2±0.3; 0.9±0.1) and Ad-NIS-CG (1.1±0.1; 0.9±0.1) compared withthe UW group (0.44±0.03; 0.47±0.06) on POD 5 and 10, respectively (%ID/g; mean ± SD)(p<0.05). Image intensity at days 5 and 10 was higher than at day 15 in the hearts transducedwith Ad-NIS, Ad-NIS-CEA and Ad-NIS-CG (Table 1) while UW group image intensity didnot differ on sequential imaging over time. A peak in image intensity on day 5 followed by afall to background level by day 15 was noted and reflects the serotype 5 recombinant adenoviruskinetics in the transplanted hearts (Figure 1).

Soluble marker hCEA and βhCG serum quantificationSerum quantification of soluble markers hCEA and βhCG was performed on blood collectedfrom all the recipients on POD 5, 10 and 15 before each 123I injection and micro-SPECT/CTimaging. In the Ad-NIS-CEA group mean ± standard deviation serum levels of hCEA were4.4±6.0, 0.9±1.1 ng/ml on POD 5 and 10 respectively. On POD 15 hCEA serum levels werebelow the threshold (0.1 ng/ml) for all the 6 animals. As for image intensity a peak level wasnoted on POD 5 followed by a progressive decrease level by day 15. hCEA levels weresignificantly higher in Ad-NIS-CEA group compared to the other three groups on days 5 and10. No difference was found between the groups on day 15. hCEA was undetectable in all thesamples obtained from UW, Ad-NIS and Ad-NIS-CG perfused hearts.

Differently from the trend observed for hCEA levels in Ad-NIS-CEA group, in the heartsperfused with Ad-NIS-CG, serum levels of βhCG were significantly higher (7.8±6.2 IU/L)only on POD 5 respect to POD 10 and 15 (p<0.05). On POD 10 and 15 βhCG serum levelswere constantly below the threshold (0.5 IU/L). In the hearts perfused with UW only, Ad-NISor Ad-NIS-CEA, βhCG was undetectable in all the serum samples analyzed. Soluble markerserum levels are listed in Table 2.

Anti-hCEA and anti-βhCG antibody screeningAnti-hCEA antibodies were detected on serum samples from Ad-NIS-CEA rats only on POD15 while they resulted negative on POD 5 and 10 (Figure 2A). Serum samples collected fromAd-NIS-CG rats on POD 5 contained no detectable anti-βhCG antibodies. In this group,differently from Ad-NIS-CEA group, on POD 10 and 15 significantly high titers of anti-βhCGantibodies were detected (Figure 2B). These findings explain the washout of serum hCEA andβhCG noticed on POD 15 and after POD 5 respectively.

Correlation between micro-SPECT imaging and soluble markers quantificationImage intensities of micro-SPECT images expressed as %ID/g were compared with hCEA andβhCG serum levels at POD 5, 10 and 15. The analysis was performed comparing %ID/g vshCEA levels in Ad-NIS-CEA perfused hearts and %ID/g vs βhCG levels in hearts perfusedwith Ad-NIS-CG. Serum levels of hCEA and βhCG correlated well with image intensities onPOD 5, R2=0.99 with p<0.001 and R2=0.96 with p=0.001 respectively (Figure 3A and 3B).On POD 10 a strong correlation was found between %ID/g and hCEA levels (R2=0.88,p=0.006) as shown in Figure 4. %ID/g and βhCG serum levels didn’t correlate on the samePOD because of βhCG serum levels constantly below the detection limit. No correlation wasfound between %ID/g and both the soluble markers levels on POD 15 (data not shown).

This result suggests that serum levels of hCEA and βhCG increased only in animals showinghigh cardiac 123I uptake, but not in those with lower uptake. Above this threshold (0.6 %ID/g), image intensities correlated well with serum levels of hCEA and βhCG. The threshold valuefor image intensity (%ID/g) was estimated from the UW only perfused hearts data pooledacross the three time points (POD 5, 10 and 15), which represented the range of baselineamount. Since this cut-off served to differentiate baseline versus virus-induced %ID/g, the

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maximum value from the baseline data plus one standard deviation was chosen as the thresholdvalue (0.6 %ID/g).

Sensitivity and logistic analysisThe sensitivity of %ID/g computed at day 5, 10, and 15 was 100%, 100%, and 16.7%respectively (Table 3A). In a logistic regression model predicting any of the three Ad-NIS, Ad-NIS-CEA and Ad-NIS-CG treatment groups vs UW group using %ID/g value, the c-statisticwas 1.0, 1.0, and 0.90, respectively. Pooling across all 3 days, the sensitivity was 72.2%, andthe corresponding logistic analysis yielded a c-statistic of 0.97 (95% CI: 0.92, 1.00), indicatingvery strong discriminatory ability.

For hCEA serum, the sensitivity was 100% at days 5 and 10, and 0% at day 15. When poolingdays together, the sensitivity was 66.7% and the c-statistic was 0.83 (95% CI: 0.72, 0.95). Asfor the other serum parameter βhCG serum level, the sensitivity at day 5 was 100% and thendropped down to 0% at day 10 and 15. Essentially, pooling across days, the overall sensitivityof βhCG serum levels was 33.3% and the corresponding c-statistic was 0.67 (95% CI: 0.56,0.78).

To compare the performance of the three parameters, we compared the c-statistics from thepooled data. Specifically, we tested whether the c-statistic for the %ID/g parameter wassignificantly higher than that of the hCEA or the βhCG serum level parameters. The ability of%ID/g of discriminating those induced with Ad-NIS, from those not, was significantly higherthan the discriminatory ability of hCEA among those with and without Ad-NIS-hCEA and ofβhCG among those with and without Ad-NIS-βhCG (p-value=0.03 and <0.001, respectively)(Table 3B).

DiscussionRecent improvements in experimental research of gene transfer suggest that gene therapy maybecome a promising choice for the treatment of rejection[12]. The success of this therapydepends on the efficiency of gene transfer with the use of a non-immunogenic and durablevector, such as adeno associated virus (AAV) (manuscript sbmitted).

In this study the heterotopic hearts perfused with a solution containing either Ad-NIS, Ad-NIS-CEA or Ad-NIS-CG presented a significantly higher uptake of radioiodine with respect to thecontrol group (UW only), 5 and 10 days after the ex vivo perfusion. Except for the last groupall transplanted hearts showed an image intensity peak on POD 5 with a radioiodine uptakethat was still significantly higher on POD 10 than in the control group. The percentage ofinjected dose per gram returned to the baseline level by day 15 revealing no difference withrespect to the controls. This trend was expected according to the kinetics of adenovirus serotype5.

It would be tempting to state that hNIS gene is more sensitive as a reporter gene than hCEAor βhCG which code for soluble marker peptides. Serum levels of soluble markers peptideincreased in animals showing increased cardiac I123 uptake, but not in those with decreaseduptake. However, the threshold value for image intensity (%ID/g) estimated from the UWgroup represents only arbitrarily the range of a baseline detection level. Again, strongcorrelation between image intensity and serum level of soluble markers was noted for bothhCEA and βhCG on POD 5 when, according to the adenovirus 5 kinetics, the maximum geneexpression is expected. Only hCEA levels were highly correlated with the radioiodine uptakesafter 10 days post transplantation while βhCG resulted undetectable in all the samples. Thisresult can be explained by the presence in serum of a high titer of anti-hCEA and anti-βhCG

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antibodies after POD 10 and 5 respectively. In addition the small number of grafts in our studyin each group obviously limits final conclusions concerning the sensitivity of these markers.

Gene therapy is a very promising type of treatment for many different diseases. In theory, genetherapy can repair the cause of genetic disorders on the most basal level and thereby be veryspecific and effective. By selectively introducing genes that encode for so called suicideproteins, even cancer can be treated with gene therapy. It has become clear that the success ofthe therapy is highly dependent on the efficiency of gene transfer with the use of a non-immunogenic and durable vector. It is also mandatory to define non-invasive method to monitorgene transduction.

Ideally, in any gene therapy study, we should know the number, distribution and fate of thegenetically modified cells, and the kinetics of production of the therapeutic protein over time.Unfortunately, for most transgenes this type of information is very difficult to obtain. Mappingthe distribution of genetically modified cells typically requires euthanasia and thorough post-mortem analysis of all tissues using in situ hybridization, immunohistochemical analysis, ordetermination of enzymatic activity in tissue extracts. Non-invasive imaging has recently beenused to map the distribution of transgene expression in one or two special cases where thetransgene product can be used to track a positron-emitting radionuclide at its site of expression[13].

Equally problematic is the need for non-invasive methods to monitor the profile of geneexpression over time. Expression of cell-associated transgenes can be monitored byimmunohistochemical or other analysis of tissue biopsies obtained from the site of gene transfer[14–16]. However, for most vector systems and most routes of administration, gene transferwithin the target site is non-uniform. Tissue sampling is therefore an unsatisfactory method toassess the efficiency of gene delivery because the biopsy that is subjected to analysis isessentially a random sample from an area of non-uniform gene delivery. Also, there is a limitto the number of times that a given tissue can be biopsied, and for certain tissues, such as theheart, tissue biopsy is associated with a high risk of fatality.

Even for secreted transgene products, the monitoring of expression can be fraught withproblems. First, the secreted protein may be retained in the tissues through a heparin-bindingor matrix attachment region or through its interaction with its naturally occurring receptor.Thus, human cardiovascular gene therapy studies, employing vectors coding for vascularendothelial growth factor, were associated with transient or undetectable circulating levels ofthe protein [17–19]. Second, the soluble protein encoded by the transgene may beindistinguishable from its naturally occurring counterpart. This is a problem in cancer genetherapy studies where immunostimulatory cytokines, such as IL-2, GMCSF and IL-12expressed from a transgene delivered in the tumor microenvironment, cannot be distinguishedfrom the same cytokines expressed from activated macrophages and lymphocytes that are partof an inflammatory response against the vector. Third, for certain proteins there may be nosuitable assay available for sensitive detection. Finally, the soluble protein may be the targetof a humoral immune response that causes a precipitous fall in the circulating level giving themisleading impression that its production is attenuated.

Our data demonstrate that hNIS is an excellent reporter gene for the transplanted heart. Theexpression level of hNIS can be accurately and non-invasively monitored by serialradioisotopic SPECT imaging. In addition hNIS gene transfer permits sequential real-timedetection and quantification of reporter gene expression in the transplanted heart. We alsodemonstrated that there is a very high concordance between imaging and soluble markerpeptides. Thus, the expression level of one gene can be inferred from the measured expressionlevel of the other.

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In myocardium NIS protein doesn’t interact with the cell biochemistry since iodide is notmetabolised. Despite there was a concern in the use of NIS because of the possible membranepotential effects in the transfected myocytes, in this study all animals were clinically stablethroughout the observation period with a preserved palpation score (6/6). Lee et al. in theirstudy demonstrated, in fact, that myocardial NIS gene imaging does not cause significantmyocardial injury or perturbed cardiac function other than mild effects due to adenoviralvector-associated host response [8]. In addition the use of a species-specific isoform such ashNIS for humans may help to circumvent any problem caused by the immunogenicity oftransgene products.

Adenovirus elicits a strong host immune response, this interferes imaging of reporter geneexpression over about ten to fourteen days after adenovirus transfection. Transient expressiondue to immunogenic reaction is a very well known limitation of this vector. Despite these limitswe choosed to use a recombinant adenoviral vector, with a deleted E1 region to preventreplication in vivo, because of the easy production, the long–term experience and the highefficiency obtained with this vector [10,20–22].

The aim of this study was to demonstrate the usefulness of hNIS as reporter gene for SPECT/CT imaging independently of the vector used for transfer gene. Moreover the use of thistechnique with a dual-gene vector appears to allow accurate assessment of the level ofmyocardial expression of a second gene of interest.

The results obtained lead to future applications of the same reporter gene encoded into lessimmunogenic and more durable vectors like recombinant AAV [23]. These vectors couldensure, for a long period, an adequate time course monitoring of transplanted cells after exvivo transduction. Recently we have made AAV vectors coding for hNIS, that unlikeadenovirus vectors they do not encode viral proteins; in this way the transduced cells are notimmunogenic. Preliminary in vivo data are looking promising.

Finally, the use of ex vivo perfusion technique is feasible in the large animal model [24]. Thismethod was not used in this study due to constraints with the production of large quantities ofthe vector. However, the translation of this technique to humans would be straightforward dueto the expertise already gained with SPECT imaging of the heart in clinical studies. The useof SPECT/CT in the transplanted human heart would also facilitate precise quantification ofcardiac dimensions and exact sites of gene transfer. Small animal SPECT/CT scanners arelimited in their resolution. Soft tissue and bowel shadows in the abdomen in this heterotopicmodel can have affected definition of transplanted heart margins.

Taken together, our data suggests that hNIS is a most promising reporter gene for thetransplanted heart. The expression level of hNIS may be monitored non-invasively by serialradioisotopic SPECT imaging. In addition, hNIS gene transfer permits sequential real-timedetection and quantification of reporter gene expression in the transplanted heart.

Acknowledgements

This research is supported by: NIH Grant HL66598, William J von Liebig Foundation

We wish to acknowledge the contributions of Dr. Kent Bailey and Brian Lahr for statistical analysis, Teresa Deckleverfor imaging assistance and Karen Schumacher for writing assistance.

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Figure 1.Micro-SPECT antero-posterior view of the abdomen of the same animal showing thetransplanted heart on POD 5 (left), 10 (middle) and 15 (right) after Ad-NIS perfusion. The “hotspot” corresponds to the position of the transplanted heart (*). The “hot spot” above thetransplanted heart corresponds to the 123I concentration in the stomach due to the inherentpresence of NIS in the gastric mucosa. The regions of interest were defined manually aroundthe heart shadows on the reconstructed axial tomographic images in all sections in which theheart was visualized, and quantification performed on the co-registered micro-SPECT images.This ensured accurate co-registration of the axial CT images with the micro-SPECT maps.

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Figure 2.Mean serum levels of hCEA (A) and βhCG (B) on POD 5, 10 and 15. The presence or absenceof anti-hCEA or anti-βhCG antibodies in the serum samples are as indicated.

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Figure 3.Comparison of image intensity %ID/g with hCEA (A) or βhCG (B) serum levels on POD 5.Dashed lines represent the thresholds of %ID/g in both A and B, hCEA in A and βhCG in B.After 5 days from ex vivo heart perfusion with Ad-NIS-CEA or Ad-NIS-CG image intensitiesand soluble markers serum quantifications correlate very well (A: %ID/g vs hCEA R2=0.99,p<0.001 and B: %ID/g vs βhCG R2=0.96, p=0.001).

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Figure 4.Comparison of image intensity %ID/g with hCEA serum levels on POD 10. Dashed linesrepresent %ID/g and hCEA thresholds. After 10 days from ex-vivo Ad-NIS-CEA heartperfusion %ID/g still correlates well with hCEA serum levels (R2=0.88, p=0.006). Nocorrelation was found on the same post-operative day between image intensities and βhCGserum levels (from POD 10 βhCG levels were constantly below the threshold).

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Table 1Image intensity quantification on POD 5, 10 and 15 following gene transfer or UW only perfusion. Data areexpressed as percentage of injected dose of 123I per gram of heart tissue (%ID/g). Image intensity was significantlyhigher in the Ad-NIS, Ad-NIS-CEA, Ad-NIS-CG than UW only perfused hearts on POD 5 and 10 (*p<0.05, t-test). No differences in radioiodine uptake between the groups were noted on POD 15 imaging comparing UWgroup with either Ad-NIS or Ad-NIS-CEA or Ad-NIS-CG group (t-test).

N POD 5 POD 10 POD 15

UW 3 0.44± 0.03 0.47± 0.06 0.45± 0.07Ad-NIS 6 1.1± 0.2* 0.9± 0.07* 0.54± 0.04

Ad-NIS-CEA 6 1.2± 0.3* 0.9± 0.1* 0.57± 0.06Ad-NIS-CG 6 1.1± 0.1* 0.9± 0.1* 0.57± 0.09


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Ricci et al. Ta

ble

2So

lubl

e m

arke

r hC

EA a

nd β

hCG

seru

m le

vel q

uant

ifica

tion

on P

D 5

, 10

and

15 fo

llow

ing

gene

tran

sfer

or U

W o

nly

perf

usio

n. D

ata

are

expr

esse

d as

mea

n±

SD. h

CEA

ser

um le

vel w

as s

igni

fican

tly h

ighe

r in

Ad-

NIS

-CEA

gro

up o

n da

ys 5

and

10

resp

ect t

o th

e ot

her t

hree

gro

ups

(*p<

0.05

, t-te

st);

in th

e sa

me

grou

p βh

CG

seru

m le

vel w

as u

ndet

ecta

ble

in a

ll th

e an

alys

is. β

hCG

seru

m le

vel w

as si

gnifi

cant

ly h

ighe

r in

Ad-

NIS

-CG

gro

up o

nly

on P

OD

5 re

spec

t to

the

othe

r thr

ee g

roup

s (*

p<0.

05, t

-test

); in

the

sam

e gr

oup

hCEA

ser

um le

vel w

as u

ndet

ecta

ble

in a

ll th

e an

alys

is. N

o di

ffer

ence

s w

ere

note

d in

ser

um le

vels

obta

ined

from

UW

and

Ad-

NIS

per

fuse

d he

ars

on s

eque

ntia

l hC

EA a

nd β

hCG

ser

um q

uant

ifica

tion

over

tim

e, in

thes

e tw

o gr

oups

bot

h hC

EA a

nd β

hCG

resu

lted

unde

tect

able

in a

ll se

rum

sam

ples

(hC

EA <

0.1

ng/m

l, βh

CG

<0.

5 IU

/L).

POD

5PO

D 1

0PO

D 1

5

NhC

EA

(ng/

ml)

β hC

G (I

U/L

)hC

EA

(ng/

ml)

β hC

G (I

U/L

)hC

EA

(ng/

ml)

β hC

G (I

U/L

)

UW

3<0

.1<0

.5<0

.1<0

.5<0

.1<0

.5A

d-N

IS6

<0.1

<0.5

<0.1

<0.5

<0.1

<0.5

Ad-

NIS

-CE

A6

4.4±

6.0

*<0

.50.

9± 1

.1*

<0.5

<0.1

<0.5

Ad-

NIS

-CG

6<0

.17.

8± 6

.2*

<0.1

<0.5

<0.1

<0.5


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Table 3A.Sensitivity of %ID/g, hCEA (ng/ml) and βhCG (IU/L) computed at POD 5, 10 and 15. B. Values representthe c-statistics in a logistic regression model predicting any of the three Ad-NIS, Ad-NIS-CEA and Ad-NIS-CGtreatment groups vs UW group. CI, confidence interval. (*) comparing c-statistic to that of %ID/gr.

Sensitivity (c-statistic)

A. POD 5 POD 10 POD 15 Total Days

%ID/g 18/18 =100% 18/18 =100% 3/18 =16.7% 39/54 =72.2%hCEA (ng/ml) 6/6 =100% 6/6 =100% 0/6 =0% 12/18 =66.7%βhCG (IU/L) 6/6 =100% 0/6 =0% 0/6 =0% 6/18 =33.3%

Total Days

B. c-statistic 95% CI p-value*

%ID/gr 0.966 (0.924, 1.000) -hCEA (ng/ml) 0.833 (0.721, 0.945) 0.03βhCG (IU/L) 0.667 (0.555, 0.779) <0.001


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