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 cells 
were seen in the spleen, liver, and in the lungs, compatible with the distribution of normal 
macrophages 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 innoculation. 
3. Toxicity studies of HS-tk VPC with and without GCV in normal brain . 
Ten Rats were inoculated with 3x1 0^ PA317/HS-tk VPC into the deep white matter of the 
cerebral hemisphere (Appendix D). The contralateral hemisphere served as control with 
3x10^ B-GAL-transduced 3T3 cells (3T3-B-GAL). Cells were injected in a volume of 50/zL. 
Rats were treated with GCV at a dose of 1 5mg/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 did not show evidence of inflammation or destructive changes in 
either group. Dexamethasone was administered to the rats as an oral dose of 0.5 mg/Kg/day, 
starting on the third day post injection of the VPC. It appeared that dexamethasone 
pretreatment diminishes the non-specific symptoms related to the surgery and implantation of 
cells in the brain. 
4. Toxicity studies in non-human primates . Five monkeys (Macaca mulata, 6-8 Kg) were used 
for the primate experiments. The study was designed to address 2 major issues: 1 . What is 
the fate (survival time, proliferation potential) of the murine VPC within the brain? 2. Is 
there significant toxicity from the intracerebral injection of HS-tk vector-producer cells, 
alone or with subsequent GCV therapy? 
As is in standard neurosurgical practice, the monkeys were treated with high-dose 
steroids (dexamethasone, 2 mg/Kg IM/day) starting 7 days before surgery and continued for 
3 weeks at which point the dose was gradually tapered during one week and discontinued. 
Antibiotic therapy (Cefotaxime, 30 mg/Kg, IM BID) was administered starting on the day ot 
surgery and continued for 10 days. Using general anesthesia (IV pentothal to effect, 
Isoflurane 0.75% - 2.0% by inhalation) under aseptic conditions, monkeys received 
stereotaxic intracerebral injection of 10^ HS-tk VPC mixed with 10 B GAL VPC (total 
Recombinant DNA Research, Volume 17 
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