Amikacin but does not inactivate other aminoglycoside antibiodcs (such as gentamicin and 
tobramycin) (26). Due to the availability of a large number of antibiotics for use in the 
treatment of gram negative infections, the NeoR gene should not affect the management of 
gram negative infections. 
F. Preparation of the GlTkSvNa.29 Vector- Producer cell line. The generation of 
retroviral vectors from transinfected PA3 1 7 cells has been extensively tested in vitro and in 
human gene transfer/therapy experiments. The GITkSvNa transinfected PA317 cells were 
selected in G418 and cloned. Clone 53 has produced the highest NeoR and HS-tk titer 
(5x10^ colony forming units/mL for both) and this clone has been used in clinical trials at 
the NIH. It has been shown to be free of detectable replication competent virus. 
G. Growth of the GITkSvNa. 29 Vector-Producer cell line for Clinical Use 
1. The producer cells are grown in complete medium which contains Dulbecco’s modified 
MEM with 4.5 gm glucose/liter with L-glutamine (DMEM), 10% FCS, 2 mM L-glutamine. 
2. Monolayer Culture (flasks and roller bottles): The monolayer cultures are grown in T-75 
flasks at 37°C with 5% C02. When the cells are >90% confluent, the cells are trypsinized 
and split 10:1 in fresh complete medium. The cultures are scaled to yield sufficient cells for 
clinical trials. A cell bank, working cell bank, and a product cell bank has been prepared by 
GTI, and have been shown to be free of detectable adventitious agents (See Appendix K). 
H. Testing and Harvest of the Producer cell line for Clinical Use 
1 . An aliquot of supernatant will be sent for a ST AT gram stain and culture. 
2. The cells will be trypsinized, harvested into 50 mL conical tubes (Falcon), washed three 
times in normal saline and counted by trypan dye exclusion. 
3. The final cell concentration will be adjusted to 1-2 xlO^ cells/mL. 
Tests done on the product lot used for clinical administration are listed in Appendix K 
V. Potential Hazards Implanting of GITkSvNa. 29 VPC into Human Brain Tumors 
A. Potential surgical complications: 
Infection: Patients with malignant brain tumors have a significant predisposition to a variety 
of superimposed infections secondary to a state of immune suppression as previously 
described. The probability of post-operative wound infection is also increased due to 
previous radiation damage to the scalp following radiotherapy. Antibiotic therapy will be 
given as a prophylaxis prior to the surgical interventions and as medically indicated. 
Increased ICP : Patients with an intracranial space occupying lesion often present with 
symptoms attributable to increased intracranial pressure (ICP). All the patients in this study 
will be treated prophylactically with high dose dexamethasone before the surgical 
procedures. Additional measures (Mannitol) may be given in the peri-operative period. 
Steroid complications : The long term effects of steroids are well documented. However, 
most of the patients who will enroll in our study will require steroids for control of increased 
ICP regardless of the proposed treatment. Inducing a reduction in tumor size may actually 
allow the reduction of the steroid dose in some patients. 
Chemical meningitis: Despite the lack of a meningeal reaction in our model laboratory 
animals, meningitis or meningitis-like symptoms may develop secondary to spillage of ^ 
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