Feasibility of sodium/iodide symporter gene as a new imaging reporter gene: comparison with<Emphasis Type="Italic"> HSV1-tk</Emphasis>

Positron emission tomography (PET) imaging reporter genes, such as HSV1-tk and D₂ receptor genes, make it possible to visualise gene expression non-invasively and repetitively in vivo. However, these systems require the synthesis of complicated substrates and the availability of expensive PET equipment. Expression of the sodium/iodide symporter (NIS) gene can be easily monitored with radioiodines and technetium-99m using a gamma camera. To evaluate the possibility of using NIS as an imaging reporter gene, we compared its characteristics with those of the conventional HSV1-tk gene. The CM cell line was made by transfecting the HSV1-tk gene into CT-26 (mouse colon carcinoma cell line). The CTN and CMN cell lines were then made by transfecting the NIS gene into CT-26 and CM. We measured the uptake of iodine-125 iodovinyldeoxyuridine ([¹²⁵I]IVDU) and ¹²⁵I to evaluate the expression of the HSV1-tk and NIS genes, respectively. Each cell line was injected into four flank sites in Balb/c mice. The biodistribution study was performed after intravenously injecting [¹²⁵I]IVDU and ¹³¹I, and ¹³¹I scintigraphy was performed for the evaluation of NIS expression. In vitro studies indicated that CTN and CMN had 40- to 79-fold and 150- to 256-fold higher uptake of ¹²⁵I than CT-26 and CM, respectively. Furthermore, CM and CMN showed 57- to 69-fold higher uptake of [¹²⁵I]IVDU than CT-26 and CTN. NIS gene expression and ¹²⁵I accumulation were found to be directly correlated (R ²=0.923), as were HSV1-tk gene expression and [¹²⁵I]IVDU accumulation (R ²=0.956). Calculated signal per unit NIS and HSV1-tk mRNA expression was 23,240±3,755 cpm and 34,039±5,346 cpm, respectively. In vivo study indicated that CTN and CMN had 2.3- and 5.8-fold higher uptake of ¹³¹I than CT-26 and CM, and 1.8- and 3.5-fold higher uptake of [¹²⁵I]IVDU than CT-26 and CTN. Scintigraphy using ¹³¹I easily visualised CTN and CMN tumours. In conclusion, the NIS gene may be viewed as an imaging reporter gene with comparable performance to the HSV1-tk gene for monitoring target gene expression.     

Comparison of [<Superscript>18</Superscript>F]FHBG and [<Superscript>14</Superscript>C]FIAU for imaging of <Emphasis Type="Italic">HSV1-tk</Emphasis> reporter gene expression: adenoviral infection vs stable transfection

Earlier studies involving comparison of different reporter probes have shown conflicting results between pyrimidine nucleosides [e.g., 2'-fluoro-2'-deoxy-1--d-arabinofuranosyl-5-iodouracil (FIAU)] and acycloguanosine derivatives [e.g., penciclovir (PCV), 9-(4-fluoro-3-hydroxymethylbutyl)guanine (FHBG)]. We hypothesized that this reported discrepancy may be related to how the reporter gene is delivered to the cells—stably transfected vs adenoviral infection. We directly compared the uptake characteristics of [¹⁸F]FHBG, [³H]PCV, and [¹⁴C]FIAU in cell culture and in vivo using an adenoviral mediated gene transfer model and stably transfected cells. We further compared the uptake of three reporter probes using both HSV1-tk and a mutant HSV1-sr39tk expressing cells to assess the optimal reporter probe/reporter gene combination. [¹⁴C]FIAU accumulation was greater than that of [³H]PCV and [¹⁸F]FHBG in control cells and in HSV1-tk stably transfected cells (P<0.001). After infection of C6 cells with AdCMV-HSV1-tk (1.5×10⁸ pfu), [¹⁸F]FHBG and [³H]PCV accumulation was significantly greater than that of [¹⁴C]FIAU (P<0.01). [¹⁸F]FHBG and [³H]PCV accumulated to a significantly greater extent than [¹⁴C]FIAU in C6-stb-sr39tk+ and AdCMV-HSV1-sr39tk infected C6 cells (P<0.001). Results from the nude mice supported the results in cell culture. [¹⁴C]FIAU led to significantly higher %ID/g in C6-stb-tk+ xenografts than [¹⁸F]FHBG (P<0.05); however, compared with [¹⁴C]FIAU, [¹⁸F]FHBG led to as high %ID/g in HSV1-tk expressing hepatocytes and to significantly greater %ID/g in C6-stb-sr39tk+ xenografts and HSV1-sr39tk expressing hepatocytes. Dynamic sequential images showed that [¹⁸F]FHBG was well retained in HSV1-sr39tk expressing cells (C6-stb-sr39tk+) for at least 4 h after injection, while it was rapidly cleared from HSV1-tk expressing cells (MH3924A-stb-tk+). [¹⁴C]FIAU accumulated in HSV1-tk stably expressing cells to a greater extent than either [³H]PCV or [¹⁸F]FHBG. However, the accumulation of [³H]PCV and [¹⁸F]FHBG in adenoviral infected C6 cells or hepatocytes was equivalent to or greater than that of [¹⁴C]FIAU. These results may be due to intracellular biochemical changes (e.g., thymidine) when cells are infected with adenovirus. For adenoviral studies, the [¹⁸F]FHBG/HSV1-sr39tk combination was shown to be more sensitive than the [¹⁴C]FIAU/HSV1-tk combination HSV1-tk.     

Non-invasive in vivo imaging with radiolabelled FIAU for monitoring cancer gene therapy using herpes simplex virus type 1 thymidine kinase and ganciclovir

An experimental cancer gene therapy model was employed to develop a non-invasive imaging procedure using radiolabelled 2'-fluoro-2'-deoxy-5-iodo-1--d-arabinofuranosyluracil (FIAU) as an enzyme substrate for monitoring retroviral vector-mediated herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) transgene expression. Iodine-131 labelled FIAU was prepared by a no-carrier-added (n.c.a.) synthesis process and lyophilised to give "hot kits". The labelling yield was over 95%, with a radiochemical purity of more than 98%. The stability of [¹³¹I]FIAU in the form of lyophilised powder (the hot kit) was much better than that in the normal saline solution. The shelf life of the final [¹³¹I]FIAU hot kit product is as long as 4 weeks. Cellular uptake of [¹³¹I]FIAU after different periods of storage was investigated in vitro with HSV1-tk-retroviral vector transduced NG4TL4-STK and parental non-transduced NG4TL4 murine sarcoma cell lines over an 8-h incubation period. The NG4TL4-STK cells accumulated more radioactivity than NG4TL4 cells in all conditions, and accumulation increased with time up to 8 h. The kinetic profile of the cellular uptake of n.c.a. [¹³¹I]FIAU formulated from the lyophilised hot kit or from the stock solution was qualitatively similar. For animal model cancer gene therapy studies, FVB/N mice were inoculated subcutaneously with the HSV1-tk(+) and tk(–) sarcoma cells into the flank to produce tumours. Biodistribution studies showed that tumour/blood ratios were 2, 3.5, 8.2 and 386.8 at 1, 4, 8 and 24 h post injection, respectively, for the HSV1-tk(+) tumours, and 0.5, 0.5, 0.7 and 5.4, respectively, for the HSV1-tk(–) tumours. Radiotracer clearance from blood was completed in 24 h and was bi-exponential. A significant difference in radioactivity accumulation was revealed among the HSV1-tk(+) tumours, the tk(–) tumours and other tissues. At 24 h p.i., higher activity retention was observed in HSV1-tk(+) tumours (9.67%±3.89%ID/g) than in HSV1-tk(–) tumours (0.48%±0.19%ID/g). After seven consecutive daily treatments with the prodrug ganciclovir, planar gamma camera imaging showed HSV1-tk(+) tumour regression at day 4, and complete tumour regression at day 7. These results clearly demonstrate that the simplified n.c.a. synthesis process developed in this study is reliable and that the [¹³¹I]FIAU product is useful for in vivo monitoring of HSV1-tk gene transfer, expression and gene therapy.     

Evaluation of F-18-labeled 5-iodocytidine (18F-FIAC) as a new potential positron emission tomography probe for herpes simplex virus type 1 thymidine kinase imaging

Objective

Herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene in combination with radiolabeled nucleoside substrates is the most widely used reporter system. This study characterized 1-(2′-deoxy-2′-[¹⁸F]fluoro-β-d-arabinofuranosyl)-5-iodocytosine (¹⁸F-FIAC) as a new potential positron emission tomography (PET) probe for HSV1-tk gene imaging and compared it with 2’-deoxy-2’-[¹⁸F]fluoro-5-iodo-1-β-D-arabinofuranosyluracil (¹⁸F-FIAU) and 2’-deoxy-2’-[¹⁸F]fluoro-5-ethyl-1-β-D-arabinofuranosyluracil(¹⁸F-FEAU) (thymidine analogues) in an NG4TL4-WT/STK sarcoma-bearing mouse model.

Methods

A cellular uptake assay, biodistribution study, radioactive metabolites assay and microPET imaging of NG4TL4-WT/STK tumor-bearing mice post administration of ¹⁸F-FIAC, ¹⁸F-FIAU and ¹⁸F-FEAU were conducted to characterize the biological properties of these tracers.

Results

Highly specific uptake of ¹⁸F-FIAC, ¹⁸F-FIAU and ¹⁸F-FEAU in tk-transfected [tk(+)] cells was observed. The tk(+)-to-tk(−) cellular uptake ratio after a 2-h incubation was 66.6±25.1, 76.3±18.2 and 247.2±37.2, respectively. In biodistribution studies, ¹⁸F-FIAC showed significant tk(+) tumor specificity (12.6; expressed as the tk(+)-to-tk(−) tumor uptake ratio at 2 h postinjection) comparable with ¹⁸F-FIAU (15.8) but lower than ¹⁸F-FEAU (48.0). The results of microPET imaging also revealed the highly specific accumulation of these three radioprobes in the NG4TL4-tk(+) tumor.

Conclusion

Our findings suggested that the cytidine analogue ¹⁸F-FIAC is a new potential PET probe for the imaging of HSV1-tk gene expression. ¹⁸F-FIAC may be regarded as the prodrug of ¹⁸F-FIAU in vivo.     

Coexpression of herpesviral thymidine kinase reporter gene and VEGF gene for noninvasive monitoring of therapeutic gene transfer: an in vitro evaluation.

Coexpression of a reporter gene and a therapeutic gene may allow for noninvasive monitoring of cardiac gene therapy. We sought to evaluate the usefulness of an adenoviral vector expressing mutant herpesviral thymidine kinase reporter gene (HSV1-sr39tk) and vascular endothelial growth factor (VEGF) 121 in independent expression cassettes (Ad4tk). METHODS: Accumulation of 14C-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FIAU) and 9-(4-18F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) as reporter probes, and secretion of VEGF into medium, were determined for Ad4tk-infected H9c2 rat cardiac cells in vitro. RESULTS: In vitro tracer uptake increased with increasing vector concentration and over time. It was comparable to cells infected with adenovirus expressing only wild-type HSV1-tk (reporter probe: 14C-FIAU) or mutant HSV1-sr39tk (reporter probe: 18F-FHBG). No significant uptake was observed in cells infected with adenovirus expressing VEGF alone. With increasing vector concentration, Ad4tk-infected cells increasingly released VEGF into medium. VEGF production correlated significantly with cellular reporter probe uptake (r = 0.93; P = 0.0003). CONCLUSION: The usefulness of a vector coexpressing HSV1-tk and VEGF for noninvasive imaging of expression of a therapeutic transgene has been demonstrated in vitro. This approach may allow for future in vivo monitoring of cardiac angiogenesis gene therapy.     

In vivo imaging of herpes simplex virus type 1 thymidine kinase gene expression: early kinetics of radiolabelled FIAU

Previous studies have shown that the herpes simplex virus type 1 thymidine kinase gene (HSV1-tk), in combination with appropriate radiolabelled substrates (e.g. [I*]-2’-fluoro-2’-deoxy-5-iodo-1-β-d-arabinofuranosyluracil; I*-FIAU, where the asterisk indicates that any of the various radioactive iodine isotopes can be used), can be used as a reporter gene forin vivo monitoring of gene transfer and expression. The aim of our study was to examine the early kinetics of I*-FIAU and the possibility of utilising iodine-123-labelled FIAU for imaging of gene expression. CMS-5 fibrosarcoma cells were transduced in vitrowith the retroviral vector STK containing the HSV1-tk gene. BALB/c mice were inoculated subcutaneously with HSV1-tk(+) and tk(-) cells into both flanks. FAU (2’-fluoro-2’-deoxy-1-β-d-arabinofuranosyluracil was radioiodinated (¹²³I, ¹²⁵I) using the iodogen method. High-performance liquid chromatography purification resulted in high specific activity and radiochemical purity for both tracers ([¹²³I]FIAU and [¹²⁵I]FIAU). Biodistribution studies and gamma camera imaging were performed at 0.5, 1, 2 and 4 h p.i. In addition, the genomic DNA of the tumours was isolated for measurement of the activity accumulation resulting from the [¹²⁵I]FIAU incorporation. Biodistribution studies 0.5 h p.i. showed tumour/blood and tumour/muscle ratios of 3.8 and 7.2, respectively, for the HSV1-tk(+) tumours, and 0.6 and 1.2, respectively, for negative control tumours. Fast renal elimination of the tracer from the body resulted in rapidly increasing tumour/blood and tumour/muscle ratios which reached values of 32 and 88 at 4 h p.i., respectively. Tracer clearance from blood was bi-exponential, with an initial half-life of 0.6 h followed by a half-life of 4.6 h. The tracer half-life in herpes simplex viral thymidine kinase-expressing tumours was 35.7 h. The highest activity accumulation (20.3%±5.7% ID/g) in HSV1-tk(+) tumours was observed 1 h p.i. At that time, about 46% of the total activity found in HSV1-tk(+) tumours was incorporated into genomic DNA. Planar gamma camera imaging showed a distinct tracer accumulation as early as 0.5 h p.i., with an increase in contrast over time. These results suggest that sufficient tumour/background ratios for in vivo imaging of HSV1-tk expression with [¹²³I]FIAU are reached as early as 1 h p.i. Received 17 July and in revised form 12 November 1999     

PET imaging of HSV1-tk mutants with acquired specificity toward pyrimidine- and acycloguanosine-based radiotracers

Purpose  The aim of this study was to create an alternative mutant of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene with reduced phosphorylation capacity for acycloguanosine derivatives, but not pyrimidine-based compounds that will allow for successful PET imaging. Methods  A new mutant of HSV1-tk reporter gene, suitable for PET imaging using pyrimidine-based radiotracers, was developed. The HSV1-tk mutant contains an arginine-to-glutamine substitution at position 176 (HSV1-R176Qtk) of the nucleoside binding region of the enzyme. Results  The mutant-gene product showed favorable enzymatic characteristics toward pyrimidine-based nucleosides, while exhibiting reduced activity with acycloguanosine derivatives. In order to enhance HSV1-R176Qtk reporter activity with pyrimidine-based radiotracers, we introduced the R176Q substitution into the more active HSV1-sr39tk mutant. U87 human glioma cells transduced with the HSV1-R176Qsr39tk double mutant reporter gene showed high ³H-FEAU pyrimidine nucleoside and low ³H-penciclovir acycloguanosine analog uptake in vitro. PET imaging also demonstrated high ¹⁸F-FEAU and low ¹⁸F-FHBG accumulation in HSV1-R176Qsr39tk+ xenografts. The feasibility of imaging two independent nucleoside-specific HSV1-tk mutants in the same animal with PET was demonstrated. Two opposite xenografts expressing the HSV1-R176Qsr39tk reporter gene and the previously described acycloguanosine-specific mutant of HSV1-tk, HSV1-A167Ysr39tk reporter gene, were imaged using a short-lived pyrimidine-based ¹⁸F-FEAU and an acycloguanosine-based ¹⁸F-FHBG radiotracer, respectively, administered on 2 consecutive days. Conclusion  We conclude that in combination with acycloguanosine-specific HSV1-A167Ysr39tk reporter gene, a HSV1-tk mutant containing the R176Q substitution could be used for PET imaging of two different cell populations or concurrent molecular biological processes in the same living subject. Financial support  This work was supported by the NIH P50 CA86438-01, R01 CA102352; grants. Technical services provided by the MSKCC Small-Animal Imaging Core Facility, supported in part by NIH Small-Animal Imaging Research Program (SAIRP), grant R24 CA83084 and NIH Center Grant P30 CA08748.     

Therapeutic gene expression in transduced mesenchymal stem cells can be monitored using a reporter gene

AimWe constructed a recombinant adenovirus construct Ad5-sr39tk-IRES-VEGF₁₆₅ (Ad5-SIV) that contained a mutant herpes viral thymidine kinase reporter gene (HSV1-sr39tk) and the human vascular endothelial growth factor 165 (VEGF₁₆₅) gene for noninvasive imaging of gene expression. The recombinant adenovirus Ad5-SIV was transfected into rat bone marrow-derived mesenchymal stem cells (MSCs), and we measured the expression of HSV1-sr39tk and VEGF₁₆₅ to evaluate the feasibility of monitoring VEGF₁₆₅ expression using reporter gene expression.MethodsThe MSCs were infected with Ad5-SIV at various levels of infection (MOI), ranging from 0 to 100 infectious units per cell (IU/cell). The mRNA and protein expression levels of the reporter and therapeutic genes were determined using real-time RT-PCR, Western blot, ELISA and immunofluorescence. The HSV1-sr39tk expression in the MSCs was also detected in vitro using a cellular uptake study of the reporter probe ¹³¹I-FIAU. Gene expression was also evaluated in vivo by micro-Positron Emission Tomography/Computed Tomography (micro-PET/CT) imaging 1 day after injecting Ad5-SIV-tranfected MSCs into the left foreleg of the rat. The right foreleg was injected with non-transfected MSCs and served as an internal control.ResultsThe real-time RT-PCR results demonstrated a good correlation between the expression levels of HSV1-sr39tk mRNA and VEGF₁₆₅ mRNA (R² = 0.93, P < 0.05). The cellular uptake of ¹³¹I-FIAU increased with increasing viral titers (R² = 0.89; P < 0.05), and in the group that received an MOI of 100, a peak value of 30.15% ± 1.11% was found at 3 hours of incubation. The uptake rates increased rapidly between 30 and 150 minutes and reached a plateau after 150 minutes. The uptake rates of ¹³¹I-FIAU by the Ad5-SIV-infected cells were significantly higher than by the Ad5-EGFP-infected cells for all time points (t = 18.43-54.83, P < 0.05). Moreover, the rate of VEGF₁₆₅ protein secretion was highly correlated with the uptake rate of ¹³¹I-FIAU (R² = 0.84, P < 0.05). The radioactivity on the micro-PET/CT images was significantly higher in the left foreleg (which received the transfected MSCs) compared with the control foreleg.ConclusionsThese results suggest that radionuclide reporter gene imaging may be used to monitor gene expression in vivo.     

In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression

PurposeTo characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer ¹⁸F-fluoromisonidazole (¹⁸F-FMISO).MethodsThree tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe ¹²⁴I-2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (¹²⁴I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between ¹²⁴I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis.Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe ¹⁸F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with ¹²⁴I-FIAU (3 h before sacrifice) and ¹⁸F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between ¹⁸F-FMISO and ¹²⁴I-FIAU on a pixel-by-pixel basis was performed.ResultsCorrelation coefficients between ¹²⁴I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, ?0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia.Correlation coefficients between ¹⁸F-FMISO and ¹²⁴I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers.ConclusionsWe have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of this model for the evaluation of radiolabeled hypoxia tracers.