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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, 599-605
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 administration 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 24well 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 phosphatebuffered 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.