clinical or histologic perturbations of the CNS were observed in 
that monkey. 
D. In Vivo Transduction with HS-tk Vectors in Mice and Rats: 
1 . Anti-tumor Effect of In Situ Injection with an HS-tk Vector- 
producer Cell Line on the In Vivo Growth of the MCA 205 Tumor in 
Mice 
In order to determine if in vivo gene transfer technique can 
promote an increased anti-tumor effect, a PA317/HS-tk producer cell 
line was used. PA3 17 /HS-tk contains a NeoR gene promoted by the 
5 1 -LTR and an SV40 promoted HS-tk gene. Figure 1 (Appendix A: 
Reprint 1) depicts the effect of GCV on tumor growth in vivo in 
mice that were injected with lxlO 6 MCA 205 tumor cells plus 2x10® 
3T3-NeoR cells, NeoR VPC, 3T3/HS-tk or PA317 HS-tk VPC. The mice 
were ear tagged, cages coded and the tumors were measured twice 
weekly with a calipers in 3 dimensions. Tumor size is expressed 
as a volume (length x width x height) . GCV treatment was initiated 
4 days after injection of the cells and continued twice daily for 
12 doses of 150mg/kg/dose . 
All tumors grew well without GCV treatment. The growth of 
those tumors injected with 3T3-NeoR or NeoR VPC were not affected 
by GCV treatment. Tumor mixed with non-producer 3T3 /HS-tk cells 
grew somewhat slower than the 3T3-NeoR cells during GCV treatment. 
However, tumors mixed with PA3 17 /HS-tk VPC regress completely 
during 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 transduced 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 (15). 
Injection of 4xl0 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 4xl0 4 9L tumor 
cells in a volume of 50 uL into the right cerebral hemisphere. 5 
days later, the same stereotaxic coordinates were used to introduce 
either saline or 3xl0 6 PA3 17 /HS-tk producer cells in 50 uL directly 
into the growing tumor. Five 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 (5) . 
E. Bystander Effect: One of the unique features of the tumor 
Recombinant DNA Research, Volume 18 
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