M.J. Welsh and A.E. Smith, RAC Application 
epithelial cells by day 21 post infection in Monkey H. No B-galactosidase activity was 
observed in a similar preparation from a control monkey (Monkey F, below). 
PCR to test for the presence of Ad2/BGal-l DNA in the brushed samples was positive in 
the right bronchus and bronchoalveolar lavage samples from the right side for 7 days. 
Conclusions 
These studies show that recombinant adenovirus can target the bronchial epithelium, as 
well as the nasal epithelium, and direct protein expression. Using a more sensitive assay 
method we detected enzyme activity in at least 10% of cells 14 days after treatment. No 
activity was detected in control animals nor in treated samples taken at later times. 
B.2.b.(4) To what extent is expression only from the desired gene (and not from the 
surrounding DNA)? To what extent does the insertion modify the expression of other 
genes? 
Since we have no evidence for integration of adenovirus DNA into the host chromosome, 
we do not expect major changes in the expression of host cell DNA resulting from 
treatment of cells or monolayers with Ad2/CFTR-1. 
Host cell shutoff 
In the course of a normal adenovirus productive infection cycle, host cell macromolecular 
synthesis is inhibited (host cell shut-off) (123). This process is known to require Elb gene 
function and consequently, we do not expect treatment with Ad2/CFTR-1 to lead to host 
cell shut-off. Perhaps the best evidence that this does not occur is the finding that 
Ad2/CFTR-1 treated CF nasal polyp monolayers that express CFTR as evidenced by 
phenotypic correction are viable for at least 2-3 weeks in culture. Presumably, even if 
some shut off does occur, it has no deleterious effect on long-term cell metabolism. 
Viral gene expression 
Reports in the literature indicate that viruses deleted for Ela or Elb have a limited ability to 
reproduce (101,102,123-126) and in Fig. 8 we showed evidence for limited DNA synthesis 
by Ad2/CFTR-1 in human cells. This implies that, although reduced, expression of viral 
genes is not completely inhibited especially at high MOI. Indeed, it was for this reason that 
we left intact the E3 region. We therefore examined expression of early and late viral 
genes by Northern Blot analysis using as controls either wild- type or defective plus wild- 
type helper infected cells. We detected transient, dose-dependent expression of E4 and L5 
transcripts in HeLa and CFPAC cells. The levels of expression were greatly reduced 
compared with wild-type infected cells (less than 0.1%). We have not attempted to detect 
viral proteins encoded by these mRNAs, but again, our experience with human nasal polyp 
monolayers suggests that no toxicity is associated with this limited viral gene expression. 
B.2.b.(5) In what percentage of cells does expression from the added DNA occur? Is the 
product biologically active? What percentage of normal activity results from the 
inserted gene? 
It is difficult to be certain of the exact percentage of cells that express the DNA, but the 
studies with the CF airway epithelia cultured on permeable filter supports suggest that it is 
possible to complement the CF chloride transport defect at an MOI of 5 PFU/cells and that 
it is possible to obtain detectable c AMP-stimulated chloride secretion with as little as 0.1 
PFU/cell. 
[898] 
Recombinant DNA Research, Volume 16 
