I 
mutants (Shenk et al. , 1980). 
To evaluate whether DNA replication occurs in human airway epithelium 
following infection with an E1 _ E3“ recombinant adenovirus vector, bronchial 
epithelial cells were recovered by brushing via a bronchoscope from nor- 
mals. Interestingly, and different from that observed with HeLa, no AvlCFl 
DNA replication was observed as a function of time or multiplicity of 
infection (up to 1000 pfu/cell) using the 32 P0<,“ DNA labeling technique 
described above. In contrast, infection with the control Ad5 virus did 
result in DNA replication (Figure 4.1-C). 
As with AvlCFl, infection of human airway epithelium with AdCFTR (MOI up to 
1000 pfu/cell) and evaluation using 32 P0 A “ DNA labeling showed no DNA repli- 
cation (Figure 4.1-D). In contrast, infection with the control Ad5 virus 
did result in DNA replication. 
The conclusion from these studies is that despite the observations of DNA 
replication in HeLa at high MOI, no viral DNA replication is detected with 
E1~E3“ adenovirus vectors of the design of AvlCFl and AdCFTR in freshly 
isolated human airway epithelial cells even at very high MOIs (1000 
pfu/cell) . To ensure that AdCFTR will not replicate in the respiratory 
epithelial cells of the individuals to be treated with AdCFTR, the replica- 
tion potential of AdCFTR will be assessed prior to therapy, with inclusion 
of the individual only if AdCFTR does not replicate in autologous respira- 
tory epithelial cells (see Section 5.4). To decrease the possibility that 
complementation may occur, individuals will be screened for the presence of 
adenovirus in the respiratory tract (see section 4.9) and measures will be 
taken to prevent adenovirus infection by isolation before and after therapy 
(see section 5.6). 
4.2 Does the Vector Express Viral Genes in Addition to the CFTR cDNA? 
From the design of the vector, and from what is known about transcriptional 
control of the adenovirus, it is reasonable to expect that some viral genes 
may be expressed following AdCFTR infection of human epithelial cells but 
at a low level. To evaluate the potential for viral gene expression HeLa 
cells were infected (100 pfu/cell) with AvlCFl or wild type virus Ad5 and 
hexon mRNA and protein (polypeptide II, L3 transcription unit) were exam- 
ined as a function of time (Figure 4.2-A, 4.2-B). Hexon was chosen because 
it is the major capsid protein representing approximately half the total 
protein content of the virus (see section 2.2.1). As expected, HeLa cells 
infected with Ad5 rapidly produced large amounts of hexon protein. In 
contrast, hexon mRNA was barely detectable in AvlCFl infected cells and 
only then if the autoradiograms were markedly over-exposed (50-fold; see 
Figure 4.2-A). Consistent with the hexon mRNA data, immunoprecipitation 
with an anti-hexon antibody of 35 S -methionine labeled HeLa cells showed a 
large amount of de novo hexon biosynthesis soon after Ad5 infection. In 
contrast, parallel studies of HeLa infected with AvlCFl showed detectable, 
but far less hexon biosynthesis with a much delayed kinetics (Figure 4.2- 
B) . The same results were observed without immunoprecipitation i.e., 35 S- 
methionine labeled HeLa cells infected with Ad5 for 36 hr previously (la- 
beling in the last 12 hr) showed a large amount of de novo hexon biosynthe- 
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Recombinant DNA Research, Volume 16 
