Experimental Study Of HSV1-tk/FIAU Reporter Gene System Cardiac SPECT Imaging

ObjectiveRadionuelide reporter gene imaging is increasingly accepted to be a noninvasiveimaging method to monitor therapeutic gene in cardiovascular disease. In the present study,the recombinant adenovirus type 5 (Ad5-tk), carrying the herpes simplex virus 1-thymidinekinase (HSV1-tk) gene was constructed and transferred into animal myocardium. Usingautoradiography (ARG) and single photon emission computed tomography (SPECT) todemonstrating the feasibility of imaging HSV1-tk gene with¹³¹I-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil (¹³¹I-FIAU) in animal models.The optimal viral titer and imaging time were also discussed as theoretic basis for furtherhuman cardiac SPECT reporter gene imaging study.Methods1. Construction of recombinant adenoviruses (Ad-CMV-HSV1-tk)The plasmid vector, pDC316-tk, and the virus vector, E1/E3-deletedreplication-defective recombinant adenovirus type 5 (Ad5-tk), carrying the HSV1-tk geneunder the transcriptional control of the cytomegalovirus (CMV) promoter, were constructedand purified in the Vector Gene Technology Company Ltd. in Beijing.2. The radioiodination of FAUUsing solid phase oxidation with Iodogen, FAU was labeled with ¹³¹I. The product,¹³¹I-FIAU, was purified on a reverse-phase Sep-Pak C-18 column and the labelingefficiency was then assayed. Moreover, the peak fractions were identified for theradiochemical purity assessment. To study the stability in vitro, the ¹³¹I-FIAU wasincubated in serum at 37℃for 4, 12 and 24 hours before the aliquots were removed for radiochemical purity analysis.3. Autoradiography study of cardiac reporter gene expression in ratsRats receiving intramyocardial injection of Ad5-tk were divided into two experimentsgroup in terms of different aims. Rats in the first set of experiments were injected with¹³¹I-FIAU at Day 1, 2, 3, 5 and 7 after transfection of 1×10⁸pfu Ad5-tk to study thefeasibility and suitable time course for reporter gene imaging. In the second group, varioustiters of Ad5-tk (5×10⁸, 1×10⁸, 5×10⁷, 1×10⁷pfu) were used to determine the threshold andoptimal viral titer needed for detection of gene expression. The control group receivedAd5-null injection in the same way to study group. All the rats were killed 24 h afterinjection of ¹³¹I-FIAU and the hearts were rapidly dissected and measured gamma counts.The rat myocardium were performed ARG to show the expression of HSV1-tk,semi-quantitative analysis derived of region of interest (ROI) was also used to assess theARG images. Reverse transcription-polymerase chain reaction (RT-PCR) andimmunohistochemical staining were used to verify the transferred HSV1-tk gene expressionin the mRNA and protein level. The relationship of ARG images with ex vivo gammacounting and mRNA level were evaluated.4. SPECT imaging of cardiac reporter gene expression in living rabbitsRabbits receiving intramyocardial injection of Ad5-tk were divided into twoexperiments group in terms of different aims. In the first set of experiments, rabbits wereinjected with ¹³¹I-FIAU 600μCi at Day 2 after intramyocardial transfection of different titerof Ad5-tk (1×10⁹, 5×10⁸, 1×10⁸, 5×10⁷ and 1×10⁷ pfu) and then the rabbits wereperformed heart SPECT imaging at hour 6, 24, 48 and 72. The rabbit intramyocardialtransfection of 1×10⁹ pfu Ad5-tk was kept imaging at hour 96 and 120. In the second group,rabbits were transferred various titers of Ad5-tk (1×10⁹, 5×10⁸, 1×10⁸, 5×10⁷, 1×10⁷pfu) todetermine the threshold and optimal viral titer needed for detection of gene expression.Then two days later, ¹³¹I-FIAU was injected through ear vein and the rabbits wereperformed heart SPECT imaging at hour 6, 24 and 48. Semi-quantitative analysis derived of region of interest (ROI) was used to assess the SPECT images. The control group receivedaseptic saline injection in the same way to study group. The second group rabbits werekilled after SPECT imaging. The hearts of animals were dissected immediately and gammacounts were measured. The rabbits myocardium were performed RT-PCR andimmunohistochemical staining to verify the transferred HSV1-tk gene expression in themRNA and protein level. The relationship of SPECT images with ex vivo gamma countingand mRNA level were evaluated.Results1. The construction and identification of recombinant adenovirusThe recombinant plasmid, pDC316-tk, was identified by PCR,restriction enzymedigestion and sequencing. The titer of packaged recombinant adenovirus, Ad5-tk, was1.1×10¹² v.p./ml, and its infection titer was 1.6×10¹⁰ IU/mL as determined by TCID50method. A replication-defective adenovirus type 5 (Ad5-null) was used as a control with thetiter of 6.3×10⁹ IU/mL.2. Radiolabeling and stabilityFAU could be labeled efficiently by solid phase oxidation with Iodogen. Theradiolabeling efficiency of ¹³¹I-FIAU was (53.82±2.05) % (n=5). After purification onSep-Pak C-18 column, the radiochemical purity of the final product ¹³¹I-FIAU was(94.85±1.76) % (n=5). The radiochemical purity of ¹³¹I-FIAU remained above 90% afterincubated in serum at 37℃for 4, 12 and 24 hours, which indicated ¹³¹I-FIAU was stable inserum.3. Autoradiography study of cardiac reporter gene expression in ratsFrom ARG images, rats injected with Ad5-tk showed significant ¹³¹I-FIAU activity inthe anterolateral wall compared with background signals seen in the control Ad5-null rats.In the first set of experiments, the highest radioactivity in the focal myocardium could beseen at Day 1, and then progressively declined with time. Although the HSV1-tk expression could be seen even 7 days after gene transferred, it was better to get the images as early asone to two days for good image quality. In the second set of experiments, the level of¹³¹I-FIAU accumulation in the transfected myocardium was typically Ad dose-dependent.From the ARG analysis and gamma counting, the threshold viral titer was 5×10⁷ pfu, andthe optimal Ad titer was 1×10⁸ pfu concerned with viral immunoreaction. ROI-derivedsemi-quantitative study on ARG images had good correlation with ex vivo gamma countingand mRNA levels from RT-PCR analysis (R²=0.9899, P＜0.01 and R²=0.954, P＜0.01respectively for different time; R²=0.9954, P＜0.01 and R²=0.9621, P＜0.01 respectively forvarious viral titers) and gamma counting and RT-PCR also correlated well with each other(R²=0.9697, P＜0.01 for different time; R²=0.9508, P＜0.01 for various viral titers) in both oftwo sets of experiments.4. SPECT imaging of cardiac reporter gene expression in living rabbitsFrom SPECT images, rabbits injected with Ad5-tk showed significant ¹³¹I-FIAUactivity in the anterolateral wall compared with background signals seen in the controlaseptic saline rabbits. RT-PCR and immunohistochemical staining verified the transferredHSV1-tk gene expression in the mRNA and protein level respectively. In the seriallyimaged up to Day 5, the optimal images quality was obtained at 1～2 days for different viraltiters. The highest radioactivity in the focal myocardium was seen at hour 6, and thenprogressively declined with time. The level of ¹³¹I-FIAU accumulation in the transfectedmyocardium was typically Ad dose-dependent in terms of different viral titers and thethreshold was 5×10⁷ pfu. The result could be setting better in the range of 1～5×10⁸ pfu bySPECT analysis and gamma counting. ROI-derived semi-quantitative study on SPECTimages had good correlation with the %ID/g from ex vivo gamma counting and mRNAlevels from RT-PCR analysis (R²=0.933, P＜0.01 and R²=0.877, P＜0.01 respectively).ConclusionsThe present study confirmed the feasibility of adenovirus as vector, HSV1-tk as reporter gene and ¹³¹I-FIAU as reporter probe for cardiac SPECT reporter gene imaging.ROI-derived semi-quantitative study on SPECT images and ARG images had goodcorrelation with the %ID/g from ex vivo gamma counting and mRNA levels from RT-PCRanalysis The optimal Ad5-tk titer for imaging is 1～5×10⁸ pfu and the optimal imaging timeis one to two days after gene transferred. Thus, the imaging of transgene expression in theheart is feasible and might be used for the noninvasive imaging of gene therapy in cardiacdiseases. It provides a theoretic basis also for further human cardiac SPECT reporter geneimaging study.