production from producer cells which contain the vector. No replication-competent 
virus has been detected during long term culture or following administration of the 
vector to animals or humans. 
The herpes simplex thymidine kinase (HS-tk) gene is a negative 
selectable marker or “suicide” gene. When a HS-tk transduced cell is exposed to 
ganciclovir (GCV), the GCV acts as a substrate for phosphorylation by HS-tk resulting 
in a triphosphate (TP) form of the drug. This phosphorylated form (GCV-TP) inhibits 
DNA polymerase and is incorporated into DNA resulting in an inability of the cell to 
proliferate. The end result is cell death for the HS-tk transduced cells (17). The NeoR 
gene is a positive selectable marker. The bacterial NeoR gene encodes for NPT II 
(neomycin phosphotransferase II), an enzyme that will protect GITKSVNa expressing 
cells from the toxic effects of G418 ( a neomycin analog). NeoR is widely used in our 
retroviral vectors and has been used in all human clinical trials to date without adverse 
effect. NPT-II inactivates the antibiotic Amikacin but does not inactivate other 
aminoglycoside antibiotics (such as gentamicin and tobramycin) (18). The introduction 
of the NeoR gene should not affect the clinical management of gram negative 
infections in the patients. 
Preparation of the GITKSVNa-Producer cell line 
The vector construct was transfected into the PA317 (ATCC CRL 9078) 
packaging line cells. The generation of retroviral vectors from transinfected PA317 
cells has been extensively tested in vitro and in human gene transfer/therapy 
experiments and in all cases has remained free of replication-competent retrovirus. 
(See I.B.3). 
The transinfected GITKSVNa cells were selected in G418 and cloned. 
this clone has been submitted for FDA approved testing for use in our clinical studies. 
Prior to the use of the GITKSVNa-producer cell lines for injection into humans, the 
cells will be required to meet FDA specifications already in use for other clinical human 
gene therapy experiments in progress. 
[810] 
Recombinant DNA Research, Volume 15 
