CNS 18 - 10 
3.2 The GITKSVNa Retroviral Vector 
Source: Genetic Therapy Incorporated, Rockville, MD. 
Pharmacology/ Virology: GITKSVNa is a retroviral vector derived from the 
Moloney murine leukemia virus (MoMLV). This vector contains a herpes 
thymidine kinase (HS-tk) gene cDNA that is transcribed from the viral LTR and 
a bacterial neomycin resistance (NeoR) gene transcribed from an internal SV40 
(simian virus 40) early promoter (LTR-HS-tk-SV-NeoR-LTR) in the G1 vector 
backbone (Genetic Therapy Inc., Gaithersburg, MD). This Gl-based vector has 
been modified for increased safety by alteration of the gag start codon to a stop 
codon, and by elimination of viral sequences needed for the formation of the 
virus. This has been shown to minimize the potential for helper virus 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. 
3.3 HS-tk Gene 
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. 
The NeoR gene is a positive selectable marker. The bacterial NeoR gene encodes 
for NPTII (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). The introduction of the NeoR gene should not affect 
the clinical management Of gram negative infections in the patients. 
3.4 Producer Cell Line 
3.4.1 Preparation of the GlTKSVNa-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 all clinically certified 
preparations are free of replication-competent retrovirus. Further details 
are included in Protocol Appendix II. 
The transinfected GITKSVNa cells were selected in G418 and cloned. 
Clone 90 has produced the highest NeoR and HS-tk titer (5 x 10 5 cfu/ml 
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