Autoradiography study and SPECT imaging of reporter gene HSV1-tk expression in heart

Abstract

Aim

To demonstrate the feasibility and optimal conditions of imaging herpes simplex virus 1-thymidine kinase (HSV1-tk) gene transferred into hearts with ¹³¹I-2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (¹³¹I-FIAU) using autoradiography (ARG) and single photon emission computed tomography (SPECT) in animal models.

Methods

HSV1-tk inserted into adenovirus vector (Ad5-tk) and adenovirus (Ad5-null) was prepared. Rats or rabbits were divided into a study group receiving intramyocardial injection of Ad5-tk, and a control group receiving Ad-null injection. In the study group of rats, two sets of experiments, time-course study and dose-dependence study, were performed. In time-course experiments, rats were injected with ¹³¹I-FIAU on Days 1, 2, 3, 5 and 7, after transfection of 1×10⁸ pfu Ad5-tk, to study the feasibility and suitable time course for reporter gene imaging. In dose-dependence study, various titers of Ad5-tk (5×10⁸, 1×10⁸, 5×10⁷ and 1×10⁷ pfu) were used to determine the threshold and optimal viral titer needed for detection of gene expression. The gamma counts of hearts were measured. The rat myocardium was analyzed by ARG and reverse transcriptase-polymerase chain reaction (RT-PCR). SPECT whole-body planar imaging and cardiac tomographic imaging were performed in the rabbit models.

Results

From the ARG images, rats injected with Ad5-tk showed significant ¹³¹I-FIAU activity in the anterolateral wall compared with background signals seen in the control Ad5-null rats. In time-course study, the highest radioactivity in the focal myocardium could be seen on Day 1, and then progressively declined with time. In dose-dependence study, the level of ¹³¹I-FIAU accumulation in the transfected myocardium declined with the decrease of Ad viral titers. From the ARG analysis and gamma counting, the threshold viral titer was 5×10⁷ pfu, and the optimal Ad titer was 1×10⁸ pfu. The ARG images in region of interest-derived semi-quantitative study correlated well with ex vivo gamma counting and mRNA levels from RT-PCR analysis. The gamma counting and RT-PCR also correlated well with each other in both sets of experiments. Both SPECT planar and tomographic images showed clear uptake of ¹³¹I-FIAU in the anterolateral wall where Ad5-tk was injected.

Conclusion

The study confirmed the feasibility of cardiac SPECT reporter gene imaging with HSV1-tk as a reporter gene and ¹³¹I-FIAU as a reporter probe. The optimal Ad5-tk titer for imaging was 1×10⁸ pfu and the optimal imaging time was 1–2 days after gene transfer. Thus, the imaging of HSV1-tk transgene expression in the heart is feasible and may be used for the noninvasive SPECT imaging of gene therapy in cardiac diseases.

Introduction

Cardiovascular disease is one of the most serious life-threatening diseases. Although the approaches of treating heart disease including pharmacological and surgical interventions have improved greatly in recent years, some patients develop an intractable form. Gene therapy as a novel approach is rapidly evolving and holds much promise as a potential treatment for cardiovascular diseases, such as heart failure and ischemia [1], [2], [3]. A lot of promising results on myocardial gene therapy were reported from animal studies and clinical trials [4], [5], [6], and at its peak, more than 20 clinical trials on cardiac gene therapy were in progress [7]. However, most of them have ceased for many reasons, including lack of good methods for accurately tracking the transferred genes. While postmortem histology is used for preclinical animal studies, it is impractical in clinical use. Therefore, there is urgent need to develop noninvasive imaging modalities to monitor gene transfer for cardiac gene therapy.

Over the past few years, advances in imaging technologies and molecular biology have allowed investigators to visualize targeted cellular processes at a molecular or genetic level [8], [9], [10]. Reporter gene imaging provides an elegant alternative method of genetic analysis that has high sensitivity, reliability and convenience [9], [10], [11], [12]. The herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) has been studied extensively as reporter gene. HSV1-tk is normally not present in the host tissue. It encodes for an enzyme catalyzing phosphorylation and thus intracellular accumulation of marker substrates. Among various substrates, radiolabeled 2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (FIAU) demonstrated high sensitivity and selectivity for the detection of HSV1-tk expression [13]. Previously, most applications were targeted towards imaging transgene expression in tumor models [14], [15], [16]. There were a few pioneering work of this reporter system on heart disease principally using positron emission tomography (PET) and dedicated small animal PET (micro-PET) [17], [18], [19], [20], [21], [22] which showed good clinical prospection. However, this method is relatively expensive, and relies heavily on the availability of the probes.

If reporter gene imaging is to become a routine clinical tool for monitoring cardiac therapeutic gene expression, a widely acceptable clinical imaging modality must be developed for monitoring cardiac transgene expression. Single-photon emission computed tomography (SPECT) is sensitive, objective, semi-quantitative or quantitative, and widely used in clinical cardiac nuclear medicine and can provide such a convenient tool to monitoring transgene expression. The initial work of Tjuvajev et al. [23], [24], [25] on radioiodine labeled FIAU proved it possible to use SPECT for gene expression imaging. The aim of the present study was to demonstrate the feasibility of heart imaging using HSV1-tk as reporter gene and ¹³¹I-FIAU as reporter probe with autoradiography (ARG) in rat models. The optimal viral titer and imaging time were also discussed as basis for SPECT imaging. Then SPECT reporter gene heart images were obtained in the live rabbit models.