II. Pre-clinical Data 
A. In Vivo Transduction with NeoR and (3-galactosidase Vectors: 
1 . In Vivo TVansduction of the NeoR Vector (LNL6) into the MCA 205 Murine 
Fibrosarcoma 
Initial studies were performed in mice to determine if tumor cells could be 
transduced in vivo. The retroviral vector LNL6, which has a titer of 1 .0x1 0 3 cfu/ml, was 
utilized for these experiments. LNL6, which contains a NeoR gene promoted by the 5’- 
LTR, is free of replication-competent retrovirus. NeoR protects mammalian cells from 
the toxic effects of the neomycin analogue G418. LNL6 vector producer cells or control 
cells not producing vector but expressing the NeoR gene (LNL6 transduced 3T3 cells) 
and MCA 205 tumor were mixed in vitro and injected subcutaneously in syngeneic 
C57BL/6 mice. The mice were inoculated with either 2x1 0 6 MCA 205 tumor cells 
alone (group 1 ), with 1x1 0 6 MCA 205 tumor cells mixed with either 1 xl 0 6 control 
NeoR expressing cells (group 2) or with 1x10 s NeoR vector producing PA317 cells 
(group 3). The cells were mixed just prior to subcutaneous injection. The mice were 
then ear tagged, cages coded and the tumors were measured twice weekly with 
calipers in 3 dimensions. Tumor size is expressed as a volume (mm 3 : length x width 
x height). 
To minimize the possibility of contamination of the recovered tumor cells 
with 3T3 and PA317 cells, we waited 4 weeks before excising the growing tumors. The 
excised tumors were minced and digested into a single cell suspension. Tumor cells 
from each group were cultured for two weeks. The cultured cells were then placed in a 
clonogenic assay (table 1). The number of colonies that grew in media without G418 
were similar in each group with a cloning efficiency of 15-20%. There were no 
colonies with resistance to G418 in groups 1 and 2, while group 3 had a mean of 
63%±15 (range 55-73) G418-resistant colonies. A functional assay for NPT activity, the 
enzyme produced by NeoR, was positive for all G418-selected tumors in group 3 
(Figure 1). 
Recombinant DNA Research, Volume 15 
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