cells grew somewhat slower than the 3T3-NeoR cells during GCV treatment as shown in 
Figure 1 (Appendix A: Reprint 1). However, the tumors with PA317/HS-tk producer cells 
completely regressed with GCV treatment. These findings suggest that the transfer of the 
HS-tk gene by in vivo gene transfer can eradicate tumors in vivo. Injection of transducer 
cells alone is insufficient for tumor eradication. 
2. Anti-tumor Effect of HS-tk Vector-producer Cell Line Implantation into Growing 9L 
Gliosarcoma in Rats 
9L is a rat gliosarcoma cell line derived from the Fisher 344 strain. This brain tumor 
model has been well characterized (20). Injection of 4X10 4 9L tumor cells into the cerebral 
white matter of a rat results in 100% lethality by 4 weeks. Therefore, we have used this 
brain tumor model to evaluate in vivo HS-tk transduction and the subsequent anti-tumor 
response to GCV treatment. Fisher 344 male rats weighing 250-350 grams were anesthetized 
and placed in a stereotaxic apparatus. On day 0, we implanted 4x10^ 9L tumor cells in a 
volume of 50/xL into the right cerebral hemisphere. 5 days later, the same stereotaxic 
coordinates were used to introduce either saline or 3x10^ PA317/HS-tk producer cells in 
50 fx\ directly into the growing tumor. 5 days later, the rats began treatment with GCV at 
150mg/kg/dose twice daily. On the 5th day (10 days since inoculation of the tumor), the rats 
brains were examined for the extent of tumor growth. The rats treated with the HS-tk VPC 
and GCV are the only animals that experienced complete macroscopic elimination of the 
tumor (14 of 14 rats) (Appendix A: Reprint 1. Figure 2). Microscopic analysis revealed 
either no evidence of tumor (11 of 14 rats) or some residual, mostly necrotic, tumor in the 
tumor bed (3 of 14 rats). There was no evidence of vasculitis or destruction of normal 
tissues due to spread of the vector. This experiment further supports the data obtained with 
the GlNaSvBg.29 vector, that this in vivo transduction method appears to be without 
significant side effects and has substantial efficacy (10). 
E. Bystander Effect. One of the unique features of the tumor rejection in the HS-tk system 
in mice is the observation that not all the tumor’s cells must contain the inserted gene in 
order to be killed by GCV. In mice given a subcutaneous tumor in which 100% of the cells 
carry the HS-tk gene, complete tumor regressions were seen in 13 of 15 animals following 
GCV treatment. Interestingly, when tumors established from cell mixtures containing 50% 
HS-tk gene- modified cells mixed with 50% wild-type unmodified tumor cells were treated 
with GCV, almost all tumors (14/15) regressed. Even in situations where the mixed tumor 
contained 90% unmodified, wild-type tumor cells mixed with only 10% HS-tk modified 
tumor cells, complete regression of the cancer was observed with GCV treatment in 9 of 15 
animals (Table 2, Appendix A: Reprint 1). 
The mechanism of this “bystander tumor kill” is not yet completely understood. This 
may involve the production of toxic triphosphates produced by the interaction of thymidine 
kinase and GCV within the tumor leading to inhibition of DNA synthesis and death of 
replicating cells. This does not seem to involve generalized non-specific cellular toxicity 
since the overlying skin and other tissues surrounding these HS-tk treated tumors are not 
[160] 
Recombinant DNA Research, Volume 17 
