M.J. Welsh and A.E. Smith, RAC Application 
B.2.b.(6) Is the gene expressed in cells other than the target cells? If so. to what extent? 
We have not examined CFTR expression in cells other than the airway or nasal epithelium 
in animals treated with Ad2/CFTR-1. 
A number of studies have shown that functional CFTR chloride channels can be expressed 
in a wide variety of cell s in c ulture without obvious deleterious effects (9,23,25,29,33- 
36,38,66). Were Ad2/CFTR- 1 to infect human cells other than the target, it is possible that 
the Ela promoter would be active and CFTR would be expressed. We have argued above. 
Section 3.4, that because the activity of CFTR is tightly regulated, expression of the protein 
need not necessarily lead to alterations in overall ion transport or cell function. We also 
described transgenic animals expressing very high levels of CFTR in lung (71) and 
mammary glands (72) in which no deleterious effects were seen. The same conclusion was 
drawn from preliminary examination of transgenic animals expressing CFTR under the 
influence of the ubiquitous 6 actin promoter. 
B.2.c. Laboratory studies pertaining to the safety of the deliverv/expression system. 
B.2.c.(l) If a retroviral system is used: (a) - (e) 
These questions do not apply to our protocol. 
B.2.c.(2) If a nonretroviral delivery system is used: What animal studies have been done to 
determine if there are pathological or other undesirable consequences of the protocol 
(including insertion of DNA into cells other than those treated, particularly germ line 
cells)? How long have the animals been studied after treatment? What tests have 
been used and what is their sensitivity? 
Studies of the adenovirus constructs have been performed with Syrian hamsters and 
primates. In addition, safety aspects are addressed with studies of virus-treated cell lines 
and studies of primary cultures of human airway epithelia grown on permeable supports. 
1. Cultured Cells 
The consequences of administering Ad2/CFTR-1 or Ad2/6Gal-l to cultured cells were 
described above. These studies provided evidence for limited early and late viral gene 
expression and limited transient viral DNA synthesis. 
As a follow up to these results, we have in some cases examined virus replication by the 
ability of culture supernatant from Ad2/CFTR-1 treated cells to plaque or to cause CPE in 
293 cells. Such experiments reveal no evidence for Ad2 vector replication, presumably 
because even if limited gene expression occurs, insufficient protein is produced to support 
assembly of progeny virions. More importantly, we also have no evidence of vector 
replication from animal experiments as described below. 
On some occasions we have detected CPE on 293 cells in assays of Ad2 vector-treated 
human cell supernatants. When characterized by restriction enzyme analysis, only wild- 
type Ad2 virus DNA was detected. We do not know the origin of the wild-type virus, it 
could have originated in the original human cells themselves, been present at the low level 
in the input virus, or, perhaps most likely, resulted from contamination within the 
laboratory. 
Recombinant DNA Research, Volume 16 
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