2. Immunological responses - A major aspect of this protocol is to evaluate the serological 
response to the patient. The patient's serum and bronchoalveolar lavage fluid will be 
evaluated for serological responses to wild type CFTR and adenoviral proteins. 
3 . Efficiency and stability of gene transfer - Bronchoscopy performed following gene 
transfer will provide an opportunity to assess gene transfer and CFTR expression. 
Transepithelial electrical potential differences will be performed at four sites within 
the transfected segment using techniques described in section II.C.6. Superficial airway 
epithelial cells will be harvested by brushing and plated in culture. These cells will be 
analyzed for CFTR protein and adenoviral protein expression using techniques of 
immunocytochemistry (see sections II.B.1 and II.B.3) Functional correction will be 
assessed in cultured cells using the functional assay described in section II.B.3. 
Transbronchial biopsy material, containing airway and airspace tissue, will be analyzed 
for CFTR expression by immunocytochemistry and in situ hybridization. 
IV. Isolation and Production of CFTR Adenoviruses 
IV A Vector Construction 
Step 1. Construction of pAdBglll. The purpose of this step was to construct a plasmid containing 
the 5' portion of Ad 5 with deletion of El sequences and a unique cloning site. Figure 17, panel A 
summarizing this plasmid construction. The plasmid pEHX-L3 contains sequences from Ad5 
spanning map units 1 to 1 6.1 . pEHX-L3 was digested with EcoRI and Bglll and a 5.2 kb 
fragment isolated, which contains the adenoviral sequences from map units 9.2-16.1 and the 
plasmid backbone (derived from pAT 153 and described in Falck-Pedersen et al., 1989). The 
adenoviral sequences from map unit 0-1 which contains the 5' inverted terminal repeat, origin 
of replication and encapsidation signal were amplified from the original pEHX-L3 using PCR to 
insert a Nhe I site immediately downstream of the EcoR I site, and a Bgl II site at the 3' end. 
This PCR fragment and the EcoR l/Bgl II 5.2 kb fragment were ligated to produce the plasmid 
pAdBglll. 
Step 2. Construction of pAd.CMV-lacZ. A complete minigene containing the CMV enhancer and 
promoter, the lacZ gene, and the SV40 late gene polyadenylation signal was cloned into the Bglll 
site of AdBglll. The source of the minigene cassette was plasmid pCMV-p (provided by Grant 
MacGregor, Baylor). A description and schematic of the construction of pCMV-p have been 
provided by Dr. McGregor. The description is presented below verbatim and the schematic is 
provided in Figure 17, panel B. 
pUC19 (Yanisch- Perron et al., 1985) was used as a backbone for the construction of pCMV-p. 
An 8mer Notl linker (New England Bioloabs # 1029) was cloned into the Smal site of pUC19. 
This destroys the Smal site, but generates in the process two Sacll sites, one immediately on 
each side of the Notl linker. This vector was designated pN. To provide a polyadenylation signal, 
a 196 bp fragment from the SV40 genome (nucleotides 2533-2729) was purified from an 
SV40 containing vector and, following the addition of BamHI linkers, this fragment was cloned 
into the unique BamHI site within pN. The fragment is orientated such that RNA polymerase 
transcribing from a promoter upstream of the Notl site passing through the Notl site and into 
the SV40 fragment will encounter the SV40 late gene polyadenylation signal (the early gene 
polyadenylation signal appears in the opposite orientation). This vector was designated pNA. A 
180 bp region of the SV40 genome containing late viral protein gene 16s/19s splice donor and 
accept signals [obtained as a Xhol-Pstl fragment from pLI (Okayama and Berg, 1983)] was 
cloned into pNA to provide the appropriate signals. This vector was designated pNAss. The 
human cytomegalovirus immediate early gene promoter and enhancer was obtained as a 619 bp 
Thai fragment from pCM5029 (Boshart et al, 1985). This was cloned into the Hindi site of 
Recombinant DNA Research, Volume 16 
[825] 
