Reporter Hung-Jen Hsieh

Non-invasive scintigraphic monitoring of gene expression in a HSV-1 thymidine kinase gene therapy model K.W.MORIN, E.E.KNAUS and L.I.WIEBE Nuclear Medicine Communication 1997, 18, Reporter Hung-Jen Hsieh

Introduction In vivo transduction of the herpes simplex virus type-1 thymidine kinase ( HSV-1 TK ) gene and subsequent administration of antiviral drugs such as ganciclovir has emerged as a promising gene therapy protocol for proliferative disorders.

HSV-1 TK is a virus encoded enzyme that has been exploited successfully as a target for nucleoside prodrug activation for the treatment of clinical herpes infections. Recently, it has been demonstrated that introduction of the HSV-1 TK gene into proliferating tissue is an effective strategy for activating nucleoside analogue, such as gancyclovir, with cytotoxic effects.

HSV-1 TK gene transfer and subsequent administrat- ion of gancyclovir has been investigated extensively as a gene therapy strategy for the treatment of a variety of experimental neoplasms and cellular proliferation following transluminal coronary angioplasty. However, since treatment failures in animal models are associated with poor gene transfer efficiency, both qualitative and quantitative assessment of gene transfer is imperative.

We now report that HSV-1 TK expression can be detected non-invasively using scintigraphy with (E)- 5-(2-iodovinyl)-2 ’ -fluoro-2 ’ -deoxyuridine (IVFRU), which becomes metabolically trapped in tumor cells transduced with the HSV-1 TK gene on a retroviral vector. Selective phosphorylation of radiolabelled IVFRU by HSV-1 TK results in elevated radioactivity in HSV-1 TK-expressing cells in vitro and in vivo relative to cells lacking the HSV-1 TK gene.

Materials and methods IVFRU is a nucleoside analogue that display potent and selective Anti-HSV-1 activity in vitro. It is also an effective substrate for HSV-1 TK. Moreover, it is resistant to phosphorylase-mediated deglycosylation, which previously hampered the development of other analogues.

Three murine cell lines (KBALB, KBALB-LNL, KBALB-STK) were used to evaluate IVFRU. Cells (1*105 of each line) were grown in each well in 24- well culture plates. [125I] IVFRU 38pmol was added to each well and incubate at 370C IN 0.5 ml Dulbecco ’ s modified Eagles medium. At varying time after exposure, the supernatants were removed, the cells rinsed with phosphate- buffered saline, and the adherent cells were then trypsinized and removed. Cellular uptake was determined by gamma counting in a Beck-mann 8000 gamma counter.

Biodistribution study were performed in syngeneic Balb/c mice. KBALB or KBALB-STK cells (1*105) were injected subcutaneously into the flank of male Balb/c mice (n=24). Day 14, groups of three animals were sacrificed at pre-determined intervals up to 8h after injection of 370 kBq of [131I]IVFRU. Radioactivity in dissected tissues of interest was quantified using a Backmann 8000 gamma counter after dissection.

Low non-target tissue uptake of radioactivity and favorable tumor:blood ratios suggested that the KBALB-STK tumors can be imaged scintigraphically with [131I]IVFRU. In all mice of the mice imaged before ganciclovir treatment, there was specific tumor uptake together with radioactivity in the bladder and thyroid, the latter indicating a small amount of deiodination.

Results and Discussion Exposure to growth medium containing [125I] IVFRU resulted in preferential uptake in cells expressing HSV-1 TK in vitro.  Biodistribution studies in Balb/c mice bearing KBALB or KBALB-STK tumors selectively accumulate radiolabelled IVFRU in vivo. The KBALB-STK tumors accumulated much more radioactivity (1.77% of the injected dose per gram of tissue) than the KBALB tumors, and a concurrent decrease in mean blood radioactivity resulted in 10-fold increase (3.0) in the tumor:blood ratio.

 Ganciclovir treatment precluded [131I ] IVFRU uptake by tumors in all animals studied (n=6). All animals that received gancyclovir for 7 consecutive days (including the animal illustrated in the figures) had complete tumor regression.

Conclusions  There is considerable interest in the mechanism of tumor regression, largely because of the observation that tumors with as few as 10% transduced cells can complete regress after ganciclovir treatment. Although the mechanism remain unclear, the contributions of transferred ganciclovir metabolites to adjacent non-transduced cells, potentiated cell-mediated immunity and tumor ischemia appear to account for tumor destruction following in situ gene therapy.

 Radiolabelled IVFRU is a diagnostic agent that can provide valuable information pertaining to the localization and extent of HSV-1 TK expression within a tumor.  The use of the HSV-1 TK gene as a reporter for viral or plasmid vectors delivering other therapeutic genes is a potential application of this management technique.