IV.B. Generation of Recombinant Ad.CB-CFTR Virus 
The recombinant CFTR adenovirus was constructed from the plasmid pAd.CB-CFTR and a 
modified adenovirus type 5 (Ad 5), dl7001, in which the majority of the E3 region (map units 
78.4-86) was deleted. 
dl7001 was kindly provided by Dr. William Wold from St. Louis. A description of this viral 
genome as provide to us is presented below verbatum. dl7001 is derived from rec700, an Ad5- 
Ad2-Ad5 recombinant which has the Ad5 EcoRI-A (map unit 0-76), Ad2 EcoRI-D (mu 76- 
83), and Ad5 EcoRI-B (mu 83-100) fragments. dl7001 retains Ad2 sequences from map 
position 76 to 78.4, and it is deleted of sequences from 78.4 to about 86. In terms of the 
nucleotide numbering for the E3 transcription unit dl7001 retains Ad2 sequence from nt -236 
to 362 (See Cladaras and Wold, 1985). The deletion extends from nt 362 in the Ad2 sequence to 
nt 3382 in the Ad5 sequence (nt 2437 in the Ad2 sequence corresponds to nt 2482 in the Ad5 
sequence). The total deletion is 2,975 bp. Because of this deletion there are no authentic E3 
AUG's or polyA sites. 
pAd.CB-CFTR was linearized by Nhe I cleavage and cotransfected with the large fragment of Cla 
l-cut dl7001 DNA into 293 cells (a human kidney cell line containing a functional El a gene that 
provides a trans-acting El a protein) to allow homologous recombination to occur, followed by 
replication and encapsidation of recombinant adenoviral DNA into infectious virions and 
formation of plaques. Individual plaques were isolated and amplified in 293 cells, viral DNA 
was isolated, and recombinant adenoviral plaques containing the human CFTR cDNA were 
identified by restriction cleavage and Southern blot analysis. One of the positive plaques was 
plaque-purified a second time, and designated Ad.CB-CFTR. This virus was propagated in 293 
cells and recovered 36-40 hr after infection by 3 cycles of freeze thawing. All viral 
preparations were purified by CsCI density centrifugation and either followed by gel filtration 
to remove CsCI for immediate use or stored at -20 °C by diluting 1 :5 into a glycerol/BSA 
solution. Titers of viral stocks were determined by plaque assay using 293 cells. 
IV.C. Sequence Analysis of Recombinant Virus 
Ad.CB-CFTR viral DNA has been isolated from the purified viral preparation obtained after two 
rounds of plague purification. Selected areas of the viral genome will be subjected to sequence 
analysis by Lark Sequencing Technologies, Inc. Sequence determination will be performed in 
compliance with FDA/GLP procedures. 
Two areas of the genome will be subjected to complete sequence analysis including 1) the 5' end 
of the genome spanning the 5' ITR, the entire minigene cassette including CMV enhancer, p-actin 
promoter, CFTR cDNA, and a portion of E2. Regions to be sequenced will be subcloned as 
overlapping restriction fragments into pBluescript II (Stratagene) and pGem5Zf (Promega). 
Nested deletion clones will be generated in both directions for each of the subclones using a 
modified exolll SI nuclease procedure. These deletion clones will be size selected to provide 
complete coverage of each strand and sequenced using the dideoxynucleotide termination 
procedure. Internal sequencing primers will be synthesized and used to close gaps between 
contigs and to fill in any single-stranded regions. We are in the process of negotiating a 
contract with Lark. Completion of the project will take approximately three months. 
IV.D. Production of Virus 
A critical part of the safety aspects of this protocol relates to the quality control and quality 
assurance associated with recombinant virus production. It is critical to demonstrate that the 
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