Neither child has demonstrated any evidence of adverse effect due 
to the genetically altered cells. 
3. Preliminary Studies 
Pre-clinical Data 
In Vivo Transduction with NeoR and B-galactosidase Vectors: 
In Vivo Transduction of the NeoR Vector (LNL6) into the MCA 205 
Murine Fibrosarcoma (See Appendix A: Reprint 1) (5, 15). 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 l.Oxlo" 6- 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 analog G418. LNL6 VPC 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. Mice were 
inoculated with lxlO 6 MCA 205 tumor cells mixed with either lxlO 6 
control 3T3-LNL6 expressing cells or with lxlO 6 LNL6 vector- 
producing PA317 cells. The cells were mixed just prior to 
subcutaneous injection. Tumors were measured twice weekly. 
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 as shown in Table 1. (Appendix A: 
Reprint 1) . The number of colonies that grew in medium without 
G418 were similar in each group with a cloning efficiency of 15- 
20%. There were no colonies with resistance to G418 in the tumor 
plus control 3T3-LNL6 expressing cell group. While the tumor plus 
VPC group had a mean of 62% + 15 (range 55-73) G418-resistant 
colonies. An assay for NPT activity, the enzyme produced by NeoR, 
was positive for all G418-selected tumors in the VPC group. In 
control mice, PA317/LNL6 producer cells alone produce a transient 
tumor, that is rejected in 7-10 days. Since the evaluation of 
tumors in the experimental group was performed at 4 weeks, it is 
very unlikely that these G418 resistant cells are the producer 
cells. The lack of G418-resistance in controls and the lack of 
LNL6 vector production by the recovered G418-selected cells 
suggests that tjie PA317/LN6 producer line is not responsible for 
the G418-resistance. 
In Vivo Transduction of the E. coli LacZ (B-qalactosidase gene) 
Vector (GIBqSvNa) into the 9L Rat Brain Tumor. To evaluate the in 
vivo transduction dynamics within brain tumors, we have used the 
GlBgSvNa.29 vector. GlBgSvNa.29 (produced by Genetic Therapy Inc., 
GTI) has a titer of 0.5-lxl0 6 cfu/ml. This vector contains NeoR 
and the E. coli derived gene LacZ which encodes for the production 
of the enzyme B-galactosidase (B-GAL) (14). The B-GAL expression 
can be detected using an X-GAL histochemical stain. Staining the 
brain with X-GAL turns B-GAL expressing cells blue. This results 
when an indolyl is liberated from X-GAL by the action of the B- 
[56] 
Recombinant DNA Research, Volume 18 
