Safety 
To address safety issues three areas were evaluated; 1) retroviral insertional 
oncogenesis, 2) in vitro studies , and 3) safety studies based on experience from the 
human clinical trials. 
Retroviral In Vivo Studies 
The following experimental data does not relate to the STK retroviral vector, it does 
relate to the effects of retroviral transduction on oncogenesis and therefore will be 
presented in this context. Studies were undertaken to analyze the effects of in vivo 
passage on retrovirally transduced cells. Rat skin fibroblasts were isolated from a 
Fischer 344 rat and expanded in tissue culture. The non-immortalized cell line was 
designated PRSF and transduced with the pG2N retroviral vector. This vector is an N2 
based vector with an LTR (long terminal repeat) promoted growth hormone cDNA and 
a SV40 promoted neomycin resistance gene (neoR). The transduced PRSF cells 
were selected in G418 for 14 days and subsequently single clones were isolated. One 
clone, C2, producing approximately 600 ng/106 cells/ 24° was isolated. The C2 
population of cells was used for implantation into hypophysectomized Fischer 344 
rats. Alginate, a mucopolysaccharide, was used to encapsulate the C2 cells prior to 
implantation and the cell alginate mixture was implanted intraperitoneally (I.P.). We 
had previously demonstrated that encapsulated cells survived and secreted growth 
hormone for up to two weeks in tissue culture. Nineteen days post-implantation at a 
time when serum growth hormone levels were undetectable, one rat was sacrificed 
and the I.P contents, consisting of a slurry mixture, were isolated and grown in tissue 
culture in the presence of G418. Two cell populations were isolated as distinguished 
by morphology as depicted in figure 26. Plate A shows the original pre-implantation 
C2 cells as compared to plate B, which was one isolated cell population (7078) 
exhibiting normal morphology, and plate C, a second population (7078T) which was 
spindly in appearance and grows in low serum (figure 30). The C2, 7078, and 7078T 
cells were all G418 resistant and produced growth hormone at approximately 600 
ng/106 cells/24 0 . The four cell populations , PRSF, C2, 7078, and 7078T, were plated 
in soft agar to assay for transformation. Virtually no colonies formed in agar from any 
of the cell populations except the 7078T cells, which had a plating efficiency of 2.8% 
(figure 31). In addition, colony formation of the 7078T cells demonstrated a larger 
colony size than the colonies formed by the non-transformed lines. These cell lines 
[590] 
Recombinant DNA Research, Volume 15 
