in the vicinity of the tumor, no evidence of non-tumor brain transduction was evident in 
either cerebral hemisphere as shown in Figure 3 (Appendix A: Reprint 2). The most 
mitotically active endothelial cells in the area of the tumor are likely to be those responding 
to neovascularization signal within the tumor. Destruction of transduced endothelial cells 
with GCV therapy within the tumor may enhance the anti-tumor activity. The non-producer 
control cells did not demonstrate transduction of either tumor cells or normal brain and they 
were not seen in the tumor or brain after 14 days. This experiment demonstrates that local 
injection of a VPC line will transduce tumor cells in vivo, that the transduced tumor 
infiltrates surrounding brain with the non-transduced tumor and that in vivo retroviral- 
mediated gene transfer does not significantly effect adjacent normal non-proliferating brain 
tissue and that the producer cells disappear after 14 days. 
B. In Vitro GCV Sensitivity of Mammalian Cells +/- Transduction with a HS-tk Vector 
The GlTkSvNa.29 vector was transduced into the cell lines listed in Table 1, Appendix 
E in vitro using filtered supernatant from confluent producer cells. The transduced cell lines 
were then selected in G418 for 7 days at 1.0 mg/ml. Only cells expressing a functional 
NeoR gene are able to survive these G418 culture conditions producing an essentially 100% 
selected population of vector transduced cells. We then evaluated the sensitivity of the 
transduced, G418-selected cells compared to the non-transduced parent cell lines. In each 
case, the HS-tk-transduced, G418 selected cell lines were markedly more sensitive to low 
concentrations of GCV. Concentrations of 0.5-5 pg/ml were inhibitory to the HS-tk- 
transduced cells in this assay. In a clonogenic assay, HS-tk positive cells were completely 
inhibited at 0.5 pg/ml. These findings confirm that HS-tk gene-containing retroviral vectors 
can effectively transduce murine and rat tumor cells and stably express both the NeoR and 
HS-tk genes resulting in 100% kill of the transduced cells in vitro when exposed to GCV. 
All in vivo studies were done using G418-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 5x10^ HS-tk VPC and 12 received 5x10^ B-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 150mg/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 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^ 
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. 
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Recombinant DNA Research, Volume 17 
