sis (immunoprecipitation is not necessary because the hexon band is dis- 
tinct on the SDS gels). In contrast, parallel studies of HeLa Infected with 
AdCFTR showed detectable, but far less hexon biosynthesis (not shown). 
Interestingly, whereas infection of freshly isolated normal human bronchial 
epithelial cells showed de novo hexon biosynthesis following infection with 
wild type (Ad5) virus, hexon biosynthesis was not detectable in parallel 
cultures of these cells infected with AvlCFl, i.e., if hexon gene expres- 
sion does occur in human bronchial epithelium following AvlCFl infection, 
it is at a very low level (Figure 4.2-C). These differences between HeLa 
and human respiratory epithelium are consistent with the observations of 
AvlCFl DNA replication (section 4.1). Similar results were observed without 
immunoprecipitation i.e., 35 S -methionine labeled freshly isolated normal 
human bronchial epithelial cells infected with the Ad5 for 36 hr previously 
(labeling in the last 12 hr) showed a large amount of de novo hexon biosyn- 
thesis (immunoprecipitation is not necessary because the hexon band is 
distinct on the SDS gels). In contrast, parallel studies of human bronchial 
epithelial cells infected with AdCFTR showed no detectable hexon biosynthe- 
sis (not shown) . 
The conclusion from these studies is that at least one viral gene (e.g., 
hexon) other than the CFTR cDNA can be expressed (e.g., in HeLa cells), but 
at low levels, and that in the true target cells for the protocol (respira- 
tory epithelial cells) , if hexon is expressed, expression must be at a very 
low level. 
4.3 Is Respiratory Infection With the Vector Associated With Transfer of 
the Exogenous Gene to the Germ Line? 
The purpose of this protocol is to assess somatic gene transfer to respira- 
tory epithelial cells to treat the respiratory manifestations of CF. The 
endogenous CFTR gene is widely expressed, with some CFTR mRNA in almost all 
organs and cells evaluated, albeit at very low levels in some (Trapnell et 
al., 1991a; Yoshimura et al . , 1991a). Expression in the gonads has been 
observed (Trezise et al . , 1991; J. Whitsett, personal communication). From 
informal studies with E1"E3~ recombinant Ad-based vectors carried out to 
date, transfer of the vector directly to an organ (e.g., liver) has not 
been associated with transfer of the exogenous gene to other organs, in- 
cluding the gonads (Jaffe et al . , 1992). Formal studies are ongoing to 
evaluate this with respiratory tract administration of AdCFTR. 
In males with CF the question of the potential for germ line transfer is 
moot because almost all are sterile (Boat et al . , 1989; Denning et al . , 
1968; Kaplan et al . , 1968). To insure that germ line transmission is not 
possible In the males in the protocol, males will have to have documented 
sterility to be included in the protocol. Infertility does occur in females 
with CF, but it is difficult to document clinically. To insure that germ 
line transmission is not possible in the females in the protocol, the 
females to be included in the protocol will have to have definite documen- 
tation of the absence of the ovaries and/or uterus (see Section 5.4). 
Recombinant DNA Research, Volume 16 
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