2. 3. 2. 2 Gene construct 
The retroviral vector construct contains p53 cDNA with its p- 
actin promoter inserted into the LNSX or the G1 vector 24 - 25 . 
2.3.2.3 Packaging 
See section 2.3. 1.3. 
2.3.2.4 Preclinical studies 
The LNSX-p53 and the DC-p53 were transduced into H322a 
(mutant p53), H358a (deleted p53), and H460a (wt p53). H322a 
cells that underwent one cycle of infection with the wtp53 
construct but without G418 selection had an over 3-fold 
reduction in proliferation compared to cells that received either 
the unmodified vector or no treatment. Two cycles of 
transduction without G418 selection resulted in a 5-fold 
reduction in proliferation (Fig. 3). A similar result was observed 
for the H358a cell line when transduced with LNSX-p53. The 
proliferation of the H460a cell line which has a wildtype p53 was 
not altered by transduction with any of the p53 retroviral 
constructs (Fig. 4). Thus, retroviral mediated gene transfer of 
wtp53 into human lung cancer cells with deleted or mutated p53 
significantly reduces the proliferation of those cells. The 
expression of the mutated p53 protein is uniform in cultured cell 
lines as detected by immunohistochemistry. In fresh lung 
tumors that express high levels of p53 protein, expression is 
detected in >90% of cells. 
A critical question is the ability of the retroviral constructs to 
transduce established tumor cells in vivo . This question was 
addressed by injecting H460a (10 5 ) cells in the mouse right 
mainstem bronchus followed 3 days later by lavage with LNSX 
retroviral supernate (10 s CFU in 0.1ml). LNSX was used so that 
the neo gene could be used as a marker for transduction. It 
was necessary to recover tumor cells for analysis so that the AS 
construct was not used. Tumors were harvested and the 
presence of the neo gene was assessed by Southern 
hybridization. The neo gene was detected in the DNA from the 
H460a cells indicating successful transduction of the retrovirus 
30 days after lavage. Although this data is encouraging, the 
model has limitations. Direct injection of endobronchial tumor is 
not possible in this model. Other sites of direct injection do not 
accurately simulate the milieu of endobronchial lung cancer. 
Thus, definitive answers concerning efficacy must be obtained 
through this clinical trial. 
Recombinant DNA Research, Volume 16 
[429] 
