nearly 12 months following cell injection, no ill-effects or any changes from baseline 
neurological status have been observed. 
d. Radiological evaluation - MRI Studies : Both GCV-treated and non-treated monkeys MRI 
scans showed the localized injection site as an isointense lesion (7 days after cell injection) 
or hypointense lesion (7 days after start of GCV) measuring a few mm in diameter with 
enhancement of the surrounding rim following IV. injection of gadolinium. No edema or 
mass effect were noted in any of the monkeys. This appearance is compatible with a local 
breakdown of the blood-brain barrier, as is expected from the cell injection alone. 
e. Histologic studies : Mild reactive gliosis was seen in the vicinity of the cell injection site 
without evidence of edema or pathological changes in neighboring brain tissue. This was 
true for both the GCV-treated and non-treated animals. Specimens stained for myelin 
showed localized demyelination limited to the injection site which did not increase in size 
when GCV was given. A few endothelial cells adjacent to the injection site showed P- 
galactosidase activity as evidence of transduction with the B-GAL vector. 
f. Complications : The only complications observed were 2 septic complications related to the 
profound immunosuppression induced in the monkeys by high-dose steroids. One monkey 
(number 1), which had undergone implantation of the venous access port into the right heart, 
developed acute bacterial endocarditis with resultant Staphylococcus aureus septicemia and 
septic shock that led to its death 3 days after cessation of the prophylactic antibiotic therapy. 
This occurred 7 days after starting GCV therapy. An MRI study that was obtained 24 hours 
before death as well as macroscopic appearance of the brain and histology showed no 
evidence of CNS-related toxicity. Another monkey, which received bilateral injections (HS- 
tk-producer on one side and B-GAL-transduced 3T3 cells on the other side) and no GCV 
therapy, died on the 21st post-operative day (one day before planned sacrifice). Necropsy 
revealed bilateral interstitial pneumonia due to cytomegalovirus as well as systemic mycosis 
(Candida albicans cultured from all harvested organs) as cause of death. Again, no 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, we used the PA317/HS-tk producer cell line. PA317/HS-tk contains a NeoR 
gene promoted by the 5’-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 1x10^ 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. Non-producer 3T3/HS-tk 
Recombinant DNA Research, Volume 17 
[159] 
