M.J. Welsh and A.E. Smith, RAC Application 
1. Virus preparation from DNA 
To generate the recombinant Ad2/CFTR-1 adenovirus, the vector pBR-Ad2-7/CFTR was 
cleaved with BstBl at the site corresponding to the unique BstBl site at 10670 in Ad2 (Fig. 
4). The cleaved plasmid DNA was ligated to BstBl restricted Ad2 DNA. Following 
ligation, the reaction was used to transfect 293 cells by the calcium phosphate procedure. 
Approximately 7-8 days following transfection, a single plaque appeared and was used to 
reinfect a dish of 293 cells. Following development of cytopathic effect (CPE), the 
medium was removed and saved. Total DNA was prepared from the infected cells and 
analyzed by restriction analysis with multiple enzymes to verify the integrity of the 
construct. Viral supernatant was then used to infect 293 cells and upon development of 
CPE, expression of CFTR was assayed by the cAMP-dependent protein kinase (PKA) 
immunoprecipitation assay (22). Following these verification procedures, the virus was 
further purified by two rounds of plaque purification. 
Plaque purified virus was grown into a small seed stock by inoculation at low multiplicities 
of infection onto 293 cells grown in monolayers in 925 medium supplemented with 10% 
bovine calf serum (details below). Material at this stage was designated a Research Viral 
Seed Stock (RVSS) and was used in all preliminary experiments. 
2. Virus Host Cell 
Ad2/CFTR-1 is propagated in human 293 cells (ATCC CRL1573). These cells are a 
human embryonal kidney cell line which were immortalized with sheared fragments of 
human Ad5 DNA. This cell line was established in the early 1970s in Leiden, Holland 
prior to widespread HIV infection in the population. The 293 cell line expresses 
adenovirus early region 1 gene products and consequently, supports the growth of El 
deficient adenoviruses. By analogy with retroviruses, 293 cells could be considered a 
packaging cell line, but they differ from usual retrovirus lines in that they do not provide 
missing viral structural proteins; rather, they provide only some missing viral early 
functions. 
Production lots of virus are propagated in 293 cells derived from the Working Cell Bank 
(WCB). The WCB is in turn derived from the Master Cell Bank (MCB) which was grown 
up from a fresh vial of cells obtained from ATCC. Because 293 cells are of human origin, 
they are being tested extensively for the presence of biological agents. The MCB and 
WCB are being characterized for identity and the absence of adventitious agents by 
Microbiological Associates, Rockville, MD. The tests to be performed on the MCB and 
WCB are summarized below. Results of these tests, and similar tests, will be reviewed by 
the FDA before beginning the clinical trial. 
3. Tests for the 293 Master Cell Bank 
Sterility - Tests for the presence of bacterial and fungal contaminants will be performed 
according to 21 CFR 610.12. 
Mycoplasma - Tests for the presence of agar-cultivable and non-cultivable mycoplasma 
will be performed according to guidelines outlined in the FDA "Points to Consider in the 
Characterization of Cell Lines Used to Produce Biologicals (1987), Attachment: 
Recommended Test Procedures for Mycoplasmas". 
Identity - The species of origin of the 293 cells will be identified by means of isozyme and 
cytogenetic analyses. 
Recombinant DNA Research, Volume 16 
[877] 
