serum-free medium. Clones will be established and lots of frozen supernate 
prepared. This packaging cell line was used in all preclinical studies. 
Figure 1: 
2. 3. 1.4 Preclinical studies 
The 2 Kb K- ras fragment (genomic exons 2 and 3) with a f-actin promoter was 
cloned into the LNSX retroviral vectors in both orientations. The £53 cONA with 
Its f-actin promoter was cloned into the LNSX retroviral vectors in both 
orientations. The LNSX-AS-K- ras was successfully packaged in the GP+envAml2 
packaging cell line. Initial titers ranged up to 10'. The constructs were then 
transduced Into H460a cells. Specific expression of K- ras AS RNA was shown by 
slot blot analysis using vector only negative controls and a f-actin probe for a 
loading control. Western blotting studies showed that expression of the K-ras p21 
protein was specifically reduced. Next the effect of multiple cycles of 
transduction on transduction efficiency was assessed. Transduction efficiency was 
assessed on a functional level (Fig. 1). H460a cells were transduced with either 
LNSX or LNSX-AS-K- ras daily for 4 consecutive days. Cells grew for 7 days without 
selection. The percent reduction In the growth fraction of the AS transduced 
cells reflects the efficiency of transduction as growth of a selected population 
of AS transduced cells does not occur during this time period. The growth of the 
unselected AS transduced cells was less than 20X at 7 days. Thus, the simple 
manipulation of exposing cells to the packaged retrovirus for 4 consecutive days 
caused a striking increase in transduction efficiency. In a subsequent experiment 
H460a cells were transduced daily for 7 consecutive days with LNSX-AS-K-ras and 
then selected for colony formation In 6418 (Fig. 2). Colonies were compared to 
H460a cells that were not exposed to selective medium. Following selection the 
efficiency of colony formation by the transduced cells was 98X. This reinfection 
strategy Is applicable to regional therapy. The low acute toxicity of the 
retroviral constructs should permit multiple treatments. Intratracheal and 
subcutaneous injections indicate that up to 3 injections can be tolerated In mice 
with no acute toxicity. It is anticipated that the residual number of 
endobronchial tumor cells can be reduced to <10' so that an excess ratio of 
retroviral particles to proliferating tumor cells can be achieved. 
Functional transduction efficiency of LNSX-AS-K- ras In H460a cells. Growth curves are shown for 
10’ cells/well seeded In 12 well plates. H460a cells were Infected by Incubation 0.5 m of viral 
supernate stock from either LNSX or LNSX-AS-K- ras (6X10* CFU/ml ) dally for 4 consecutive days In 
the presence of 8^/ml of polybrene. The parental H460a cells served as a control. Cells were not 
selected with 6418. Cells were counted daily. The mean ± SE is shown for 3 replicates. 
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Recombinant DNA Research, Volume 19 
