All in vivo studies were done using G4 18-selected VPC and control 
non-producer cell lines that had similar patterns of GCV 
sensitivity conferred by a transferred HS-tk gene. 
C. Toxicity studies: 
1 . Assessment of HS-tk producer toxicity in the peritoneal cavity and 
in the lung. We have injected PA317/HS-tk VPC and control PA317/B- 
GAL VPC IP into mice (Appendix C) . Twelve mice received 5xl0 6 13- 
GAL VPC. The mice were observed for 7 days during which no 
evidence of toxicity was observed in either group. GCV was then 
administered at a dose of 150 mg/Kg BID for six days. During and 
after GCV administration, no toxic side effects were observed. Six 
mice in each group were sacrificed at the end of the GCV treatment. 
No gross or microscopic pathology was seen in various abdominal 
organs. The remaining 6 mice per group have been followed for >12 
months with no evidence of long-term toxicity. Next, 12, mice each 
received, 5x10 s B-GAL or HS-tk VPC IV via the lateral tail vein to 
evaluate possible toxicity to the lungs where the cells are 
trapped. No evidence of toxicity was observed before, during and 
following GCV administration. Review of microscopic slides of the 
lungs revealed no areas of necrosis or other pathology in 
comparison to the control group. 
2 . Assessment of Transduction of Non Tumor Proliferative Tissues . 
Using the B-GAL gene as a marker, rat 9L brain tumors were injected 
with the PA317/GlBgSvNa. 29 VPC. Organs were harvested and stained 
with X-GAL in order to estimate the frequency of vector expressing 
cells. Organs evaluated included the heart, lungs, liver, spleen, 
kidney, and small and large bowel. Organs were examined at 5,9, 
and 14 days following the intra-tumoral injection of the vector. 
Organs from rats in which control non-producer B-GAL cells were 
injected into their tumors served as controls. No X-GAL positive 
cells were seen in the heart and kidney. Occasional X-GAL positive 
cell were seen in the spleen, liver, and in the lungs, compatible 
with the distribution of normal macrophage which can produce 
positive results in this assay. No difference was observed between 
the frequency of X-GAL positive cells in these organs between the 
rats injected with the VPC and those injected with the non- 
producer cells. In normal bowel, there are a large number of X- 
GAL positive cells within the villi of both the small and large 
bowel. Again, no significant difference was observed between the 
non-producer and the VPC treated rats. These findings are 
consistent with the histology and suggest no significant spread of 
the vector takes place beyond the normal brain from the site of 
inoculation. s 
3 . Toxicity studies of HS-tk VPC with and without GCV in normal brain. 
Ten rats were inoculated with 3xl0 b PA3 17 /HS-tk VPC into the deep 
white matter of the cerebral hemisphere (Appendix D) . The 
contralateral hemisphere served as control with 3xl0 6 B-GAL- 
transduced 3T3 cells ( 3T3-B-GAL) . Cells were injected in a volume 
of 50uL. Rats were treated with GCV at a dose of 15 mg/Kg BID for 
7 days and sacrificed 3 days later for histological evaluation of 
the brain. The deep injection site was evident in both hemispheres 
with local changes secondary to the injection of cells. No 
difference was seen between the HS-tk VPC injection site and the 
B-GAL transduced 3T3 cell injection site. Surrounding brain tissue 
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Recombinant DNA Research, Volume 18 
