Imaging expression of adenoviral HSV1-tk suicide gene transfer using the nucleoside analogue FIRU

Abstract

Substrates for monitoring HSV1-tk gene expression include uracil and acycloguanosine derivatives. The most commonly used uracil derivative to monitor HSV1-tk gene transfer is 1-(2-fluoro-2-deoxy--D-arabinofuranosyl)-5-[*I]iodouracil (fialuridine; I*-FIAU), where the asterisk denotes any of the radioactive iodine isotopes that can be used. We have previously studied other nucleosides with imaging properties as good as or better than FIAU, including 1-(2-fluoro-2-deoxy--D-ribofuranosyl)-5-[*I]iodouracil (FIRU). The first aim of this study was to extend the biodistribution data of 123I-labelled FIRU. Secondly, we assessed the feasibility of detecting differences in HSV1-tk gene expression levels following adenoviral gene transfer in vivo with 123I-FIRU. 9L rat gliosarcoma cells were stably transfected with the HSV1-tk gene (9L-tk+). 123I-FIRU was prepared by radioiodination of 1-(2-fluoro-2-deoxy--D-ribofuranosyl)-5-tributylstannyl uracil (FTMRSU; precursor compound) and purified using an activated Sep-Pak column. Incubation of 9L-tk+ cells and the parental 9L cells with 123I-FIRU resulted in a 100-fold higher accumulation of radioactivity in the 9L-tk+ cells after an optimum incubation time of 4 h. NIH-bg-nu-xid mice were then inoculated subcutaneously with HSV1-tk (-) 9L cells or HSV1-tk (+) 9L-tk+ cells into both flanks. Biodistribution studies and gamma camera imaging were performed at 15 min and 1, 2, 4 and 24 h p.i. At 15 min, the tumour/muscle, tumour/blood and tumour/brain ratios were 5.2, 1.0 and 30.3 respectively. Rapid renal clearance of the tracer from the body resulted in increasing tumour/muscle, tumour/blood and tumour/brain ratios, reaching values of 32.2, 12.5 and 171.6 at 4 h p.i. A maximum specific activity of 22%ID/g tissue was reached in the 9L-tk+ tumours 4 h after 123I-FIRU injection. Two Ad5-based adenoviral vectors containing the HSV1-tk gene were constructed: a replication-incompetent vector with the transgene in the former E1 region, driven by a modified CMV promoter, and a novel replication-competent vector with the HSV1-tk gene in E3 driven by the natural E3 promoter. The human glioma cell lines U87MG and T98G were infected with a multiplicity of infection (m.o.i.) of 10. Forty-eight hours later the cells were incubated with 123I-FIRU and radioactivity was measured in a gamma counter. We found significantly higher levels of radioactivity in both cell lines following infection with the replication-competent vector (P<0.001). NIH-bg-nu-xid mice were then inoculated subcutaneously with U87MG cells. Tumours (approximately 1,000 mm3) were injected with 108 and 109 Infectious Units (I.U.) of either vector. After 48 h, the tracer was injected, followed by gamma camera imaging and direct measurement of radioactivity in the tumours at 4 h p.i. Images and direct measurements indicated increased uptake of tracer with higher I.U. and also demonstrated increased accumulation of tracer in the tumours treated with the replication-competent adenoviral vector (P=0.03). These results demonstrate that 123I...

Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo

“…Because killing of animals is required for TCID50 analysis, this system would reduce the number of required test animals, which would be ethically and economically beneficial. Although other imaging approaches may be useful to obtain data complementary to this approach, [21][22][23] there is unfortunately a paucity of methods with wide applicability without the need for constructing novel constructs. Because an imaging moiety cannot be easily genetically included in all oncolytic agents due to genome size, packaging, safety and efficacy issues, this coinfection strategy might be frequently useful as it is theoretically combinable with any oncolytic adenovirus.…”

Scintigraphic Imaging of HSVtk Expression in Gene Therapy

“…Radiolabeled FIRU selectively accumulated HSV-infected cells [40] and in stably transfected HSVtk-expressing cells [36,39], but not in control cells. FIRU could also detect HSVtk expression in human glioma cells after adenoviral transfection [39]. Significantly more [ 123 I]FIRU accumulated in cells that were transfected with a replication-competent adenoviral vector, than in cells that were transfected with a replicationincompetent adenovirus.…”

“…However, FIRU is selectively phosphorylated by HSVtk and therefore can be used as an imaging agent. FIRU has been labeled with several iodine isotopes by radioiodination of the stannyl precursor [39]. Radiolabeled FIRU selectively accumulated HSV-infected cells [40] and in stably transfected HSVtk-expressing cells [36,39], but not in control cells.…”

“…FIRU has been labeled with several iodine isotopes by radioiodination of the stannyl precursor [39]. Radiolabeled FIRU selectively accumulated HSV-infected cells [40] and in stably transfected HSVtk-expressing cells [36,39], but not in control cells. FIRU could also detect HSVtk expression in human glioma cells after adenoviral transfection [39].…”

Mechanisms of thymidine kinase/ganciclovir and cytosine deaminase/ 5-fluorocytosine suicide gene therapy-induced cell death in glioma cells

“…Glioblastoma is a highly lethal neoplasm and has become a candidate tumor for the development of suicide gene therapy (Oldfield et al, 1993;Kramm et al, 1995;Nanda et al, 2001;Rainov and Kramm, 2001). As the knowledge of the molecular events of TK/GCV-and CD/5-FC-mediated cytotoxicity and the mechanisms regulating sensitivity or resistance of tumor cells may be critical to improve their efficacy, we investigated and compared their mechanisms of action in glioma cells harboring either wild-type or mutant p53.…”