virus was propagated in 293 cells and recovered 36-40 hr after infection by 3 cycles 
of freeze thawing. All viral preparations were purified by CsCl density 
centrifugation and either followed by gel filtration to remove CsCl for immediate 
use or stored at -20 °C by diluting 1:5 into a glycerol/BSA solution. Titers of viral 
stocks were determined by plaque assay using 293 cells. 
III. C. Sequence Analysis of Recombinant Virus 
Sequencing of pAd.CB-CFTR (plasmid) and adenovirus Ad.CB-CFTR (viral 
genome) 
a. Using a primer to the moloney envelope sequence and then extending 
with a primer to the SV40 polyA sequence, a sequence of 434 bases has been 
generated. This sequence reads into the E2 region of both the plasmid and the 
adenovirus clone. There is 100% homology based on published sequence for the 
adenovirus 5 genome extracted from GenBank. 
b. Using a primer to a region within the CFTR gene (starting at position 
3850), a sequence of 204 bases has been generated for both clones. There is 100% 
homology based on the published sequence for the human CFTR mRNA extracted 
from GenBank. The sequence of the entire coding region of CFTR was confirmed 
in the precursor plasmid. 
c. Using a primer to the parental vector of pAd.CB-CFTR, a sequence was 
generated from the EcoRI cloning site through the 5’ end of the adenovirus 5 
genome including the 5’ inverted terminal repeat. A sequence of 282 bases was 
generated and based on published sequence for the 5’ end of the adenovirus 
genome extracted from GenBank, there was 100% homology. Sequence using the 
same primer to the adenovirus clone is ongoing; final results are anticipated in early 
February, 1993 and will be forwarded when available. 
IV. PRE-CLINICAL STUDIES 
IV. A. Efficacy 
IV.A.1. Experience with transfection of foreign genes in airway epithelia: 
The first studies transducing foreign genes into human airway epithelial cells 
were performed in 1988 (78). In these studies, primary cultures of airway epithelial 
cells were electroporated in solutions with plasmids containing the neomycin 
resistance (Neo^) gene. These studies showed that gene transfer could be effected 
by this technique but with exceedingly low frequency. Subsequently, gene targeting 
conjugates, containing transferrin complexed to plasmid DNA, were used to 
transduce airway epithelia (79). Again, gene transfer to human airway epithelial 
cells could be effected, but the efficiency was low. Finally, retroviruses have been 
used to effect gene transfer to human airway epithelial cells. Retroviral-mediated 
gene transfer efficiency can be quite high in vitro due to the large number of 
replicating cells. However, gene transfer in vivo has been of low efficiency because 
of the low cell proliferative rate of the normal airway epithelium (80-82). For these 
reasons, studies were initiated using recombinant adenovirus, which can 
theoretically achieve high efficiency gene transfer in non-dividing airway epithelial 
cells. 
Recombinant DNA Research, Volume 17 
[453] 
