title: 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-FIRU in combination with HSV1-tk is a valuable tracer for in vivo monitoring of adenoviral gene transfer.
authors.affiliation: Department of Neurology, Daniel den Hoed Cancer Centre, University Hospital Rotterdam, The Netherlands.
authors.forename: Dharmin
authors.initials: D
authors.lastname: Nanda
authors.forename: Marion
authors.initials: M
authors.lastname: de Jong
authors.forename: Ronald
authors.initials: R
authors.lastname: Vogels
authors.forename: Menzo
authors.initials: M
authors.lastname: Havenga
authors.forename: Maarten
authors.initials: M
authors.lastname: Driesse
authors.forename: Willem
authors.initials: W
authors.lastname: Bakker
authors.forename: Magda
authors.initials: M
authors.lastname: Bijster
authors.forename: Cees
authors.initials: C
authors.lastname: Avezaat
authors.forename: Peter
authors.initials: P
authors.lastname: Cox
authors.forename: Kevin
authors.initials: K
authors.lastname: Morin
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Naimi
authors.forename: Edward
authors.initials: E
authors.lastname: Knaus
authors.forename: Leonard
authors.initials: L
authors.lastname: Wiebe
authors.forename: Peter Sillevis
authors.initials: PS
authors.lastname: Smitt
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001086:Arabinofuranosyluracil / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D014408:Biomarkers, Tumor / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D005910:Glioma / Q000000981:diagnostic imaging* / Q000235:genetics / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes / Q000493:pharmacokinetics; D051379:Mice; D009705:Nucleosides / Q000493:pharmacokinetics; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D012680:Sensitivity and Specificity; D014018:Tissue Distribution; D014161:Transduction, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor; D007457:Iodine Radioisotopes; D009705:Nucleosides; D019275:Radiopharmaceuticals; D001086:Arabinofuranosyluracil; C043457:fialuridine