of animals that received ADV-tk and GCV (100 mg/kg) treatment (Fig. 7B). Instead, 
macrophages, lymphocytes, neutrophils, necrosis and hemorrhage were apparent in the 
tumor injection area (Fig. 8). Although the ipsilateral intraventricular ependymal cell lining 
appeared damaged in some specimens, no necrosis, loss of neurons, demyelination, or 
inflammatory response was observed beyond the tumors or injection sites. 
Effect of GCV Dose on ADV-tk Treatment of 9L Tumors 
The effect of GCV dosage on the effectiveness of ADV-tk treatment was determined 
by establishing 7 experimental groups (n=4 for each group) that were implanted with 9L 
cells, treated with 1 .2 x 10 9 ADV-tk and then treated with GCV at doses of 0, 10, 20, 50, 80, 
100 and 150 mg/kg twice daily for 6 days. Twenty days after tumor induction the animals 
were perfused with fixative, the brains sectioned and stained, and the tumor size measured. 
Large tumors were present in animals treated with ADV-tk and saline and in animals treated 
with ADV-(3gal and GCV whereas animals that were treated with ADV-tk and GCV at doses 
of 80 to 150 mg/kg had no residual tumors. Although animals treated with GCV at doses of 
less than 80 mg/kg had small residual tumors, morphometric analysis of tumor size in 
animals treated with different doses of GCV showed that even at lOmg/kg of GCV the ADV- 
tk plus GCV treatments had significant effects (P<0.005) on tumor size (Fig. 9.) A significant 
reduction (P>0.005) in tumor size was present in animals treated with ADV-bgal and 150 
mg/kg GCV compared to animals treated with ADV-tk and saline. This suggests that GCV 
itself, at high concentrations, may exhibit some inhibitory effects on the rate of tumor growth 
independent of thymidine kinase activity. 
Survival After ADV-tk/GCV Treatment of 9L Tumor-bearing Fischer Rats 
To test the effect of ADV/RSV-tk and GCV treatment on long-term survival, a total of 
16 tumor-bearing animals were treated with ADV/RSV-tk and GCV and 7 animals were 
treated with ADV/RSV-bgal and GCV in three experiments (Fig 10). The animals were 
monitored daily and, if they exhibited signs of morbidity or if they died, their brains were 
removed for histological analysis if possible. All control animals that were treated with 
ADV/RSV-bgal and GCV died within 22 days after tumor injection and had large intracranial 
tumors upon necropsy. In the first experimental group one animal of 5 developed a large 
extra-cranial tumor and was perfused at 79 days. Histological analysis indicated a tumor 
contralateral to the original injection site. Another animal died at 131 days of suspected 
peritonitis. The remaining 3 have lived past 210 days. In the second experimental group of 7 
animals 2 died 38 and 62 days after tumor cell injection of unknown causes. A third died at 
151 days of suspected peritonitis. To date, the remaining 4 animals have survived over 175 
days. In the last group of experimental animals 3 of the 4 animals survived over 123 days. 
One animal was perfused at 123 days and the other two were perfused at 137 days after 
tumor cell injection. No tumor cells were seen in the brains of these 3 animals. One animal 
died at 100 days. No tumor was present in the brain of this animal and no other cause of 
death was apparent at necropsy. 
V. VECTOR SAFETY TESTING IN NON-HUMAN PRIMATES 
In May 1994 we began FDA-suggested toxicity tests of the ADV/RSV-tk and GCV 
treatment in baboons ( Papio cynocephalus) in collaboration with Dr. Dee Carey and 
colleagues at the Southwest Foundation for Biomedical Research in San Antonio, TX. 
Recombinant DNA Research, Volume 20 
[727] 
