measured by the above assays, are very unlikely to generate replication competent 
virus themselves. However, as described in this protocol below, these transduced 
cells can also be monitored for a few weeks after transduction, a period sufficient to 
detect replication competent virus by the above assays. Thus monitoring transduced 
cells for a few weeks in vitro before injection into a patient will greatly reduce the risk 
of generating replication competent virus. This proposal will be designed such that the 
transduced ovarian tumor cells will be monitored for at least four weeks post- 
transduction before these cells are injected into a patient. 
3.4 Predinical studies 
In vitro and in vivo studies 
Tumor cells expressing the HSV-TK gene are killed in vitro and in vivo by the 
drug ganciclovir. HSV-TK expressing tumor cells can affect nearby HSV-TK negative 
tumor cells when exposed to GCV. The ability to kill tumor cells in vivo may affect 
preexisting tumor cells in two ways: 1) transfer of the HSV-TK phenotype from the 
HSV-TK positive cells to the HSV-TK negative cells, thus rendering the HSV-TK cells 
susceptible to ganciclovir therapy and 2) generation of host immunity against the 
killed tumor cells. The studies presented in this section will address both issues, with 
the main focus being on the former. 
Initial studies analyzed the ability of HSV-TK transduced tumor cells to respond 
to ganciclovir (GCV) in vitro. A murine fibroblast cell line. NIH 3T3, was transduced 
with either the STK or uTK, a neo^ and HSV-TK containing retorviral vectors (25). 
These transduced cells were selected in G418, a neomycin analog toxic to 
mammalian cells. A control population of NIH 3T3 cells was transduced with the LNL 
retroviral vector( provided by Dr. A.D. Miller), a neo^ containing retroviral vector, and 
selected in G418. . The cells were then placed in varying concentrations of GCV for 10- 
14 days at which time live cells (colonies) were counted. Cells expressing the herpes 
simplex thymidine kinase gene (HSV-TK) are susceptible to the ckug ganciclovir 
(GCV). GCV is a nucleotide analog which is phosphorylated by HSV-TK with a 2-3 
log efficiency as compared to the cellular thymidine kinase. The phosphorylated 
compound can then be di- and triphosphorylated by cellular enzymes. The 
triphosphate form of GCV is toxic to the cell by either functioning as a DNA polymerase 
inhibitor or a DNA chain terminator or both. There is no obvious variation between the 
ability of GCV to kill either the STK or uTK transduced cells, while control cells were 
not affected by GCV (figure 3). 
We next transduced a murine fibrosarcoma cell line, kbalb, with the STK, uTK, and 
LNL retroviral vector. Almost all HSV-TK positive cells were killed by >0.5 uM GCV, 
while the untransduced or LNL transduced kbalb cells were virtually unaffected at 
GCV concentrations of 50 uM (figure 4). The most colonies which could be counted on 
a plate is 150. Therefore, confluent plates which contain too many colonies to count 
will be scored as 150 colonies. These studies were repeated with human tumor cell 
lines, SK-N-SH and HCT (neuroblastoma and colon carcinoma), with the same results 
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