injured while the tumors expressing the genes and the admixed wild-type tumor cells are 
completely destroyed. 
The 9L rat gliosarcoma and the human glioblastoma cell lines U251 and A 172 were also 
sensitive to this “bystander" effect in an in vitro mixing experiment. GlTkSvNa.29 
transduced and non-transduced tumor cells were mixed at different ratios in 96 well 
microtiter dishes. GCV was added to the wells and 24 or 48 hours later, the cultures were 
pulsed with tritiated thymidine. These bar graphs depict a greater decrease in proliferation 
than would be expected at a GCV level of 5 . 1 fig/mL in the medium (the numbers over the 
bars represent the percent of transduced tumor in the culture) (Appendix B: Figure 1). The 
5. 1 fig/mL concentration is easily within the therapeutic range established in humans with a 
dose of 5 mg/kg/dose. Since it is unlikely that 100% of the tumor cells in the brains of our 
patients will be successfully gene-modified, this “bystander” effect is very important for the 
successful elimination of the tumor using this approach (See Appendix B: Figure 1). 
Analysis of human melanoma, colorectal, breast, lung and renal cell carcinoma cell lines 
reveal that all demonstrate the "bystander" effect in vitro. 
Pre-Clinical Summary: The pre-clinical findings noted above suggest that the direct inoculation 
of a brain tumor with HS-tk retroviral vector-producing cells can mediate complete tumor 
rejection. This in vivo gene transfer methodology selectively alters the sensitivity of a tumor 
cell to chemotherapy. Treatment with GCV does not result in widespread damage to the host 
immune system like many forms of chemotherapy. Our animal studies have demonstrated no 
significant toxicity to normal brain or any of the proliferating non-CNS tissues, suggesting 
that the implantation of VPC in a brain tumor is not associated with non-specific systemic 
toxicity. 
II. Clinical Data 
One patient has been treated with the stereotaxic injection of HS-tk VPC and GCV infusion. 
No evidence of toxicity due to cell implantation or GCV administration has been observed as 
of Jan 4, 1993. 
4. Research Design and Methods 
I. Selection of Patients 
A. Inclusion Criteria 
1. Adults with malignant brain tumors (primary and metastatic) who failed standard therapy 
for their disease will be eligible to enter the study . 
2. Patients must have surgical accessible lesions as estimated from the pre-treatment 
radiological evaluation. These decisions will be made by the Pis in accordance with the 
standards of care of neurosurgical practice. 
3. Male or non-pregnant female patients >_ 18 years of age. Women of child bearing 
potential must practice strict birth control to prevent pregnancy for the duration of the study. 
Men will be advised to use barrier protection for the duration of the study. 
4. Possess the ability to give informed consent and express a willingness to meet all the 
expected requirements of the protocol for the duration of the study. (Appendix L) 
Recombinant DNA Research, Volume 17 
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