Techniques of in situ hybridization and immunocytochemistry were used to characterize the 
distribution of CFTR expression in bronchus from non-CF and CF tissues [Engelhardt et al., 
1992b]. Figure 1 summarizes our findings in non-CF tissue. The predominant site of CFTR 
RNA and protein expression in non-CF bronchus was the submucosal glands with the highest 
expression detected in the ducts and serous tubules although low levels were detected in surface 
epithelia. Similar analyses performed on bronchi obtained from CF patients demonstrated a 
distribution of CFTR RNA expression similar to that found in non-CF tissue except there was 
markedly increased RNA expression in the collecting ducts. Expression of CFTR protein was a 
function of genotype where the most common mutation, AF 508, is associated with markedly 
decreased levels of protein in all structures, and the less common mutation, G551D allele, is 
associated with normal distribution of apicaliy localized protein. A paper describing this work 
is provided in Appendix B. 
Similar techniques were used to localize CFTR in the distal airway. These studies demonstrated 
high levels of CFTR in a subpopulation of surface epithelial cells (approximately 10-40% of 
the total) in most noncartilagenous airways (data not shown). 
These studies have identified a large number of possible cellular targets for CFTR gene transfer. 
Functional analysis and localization studies suggest that essentially all of the cells of the 
proximal surface epithelium and a substantial number of distal airway epithelial cells express 
CFTR in non-CF tissue and therefore should be targeted in gene therapy. Fortunately, these cell 
types are easily accessible by inhalation or lavage. CFTR-expressing cells within the 
submucosal glands will be more difficult to target. The current protocol, which is designed to 
reconstitute CFTR in proximal and distal surface epithelia, is based on the premise that 
correction of the submucosal is not necessary for some level of efficacy. 
II.B.2. Production of recombinant CFTR adenovirus 
Recombinant adenoviruses were generated that express a variety of CFTR alleles. Detailed 
descriptions of the methods used to construct the vectors and produce the viruses are provided 
in Section IV. 
The recombinant virus used in this protocol, Ad.CB-CFTR, is based on Ad5. In this vector, 
sequences spanning the El region from 1 to 9.2 mu are deleted and replaced with a minigene 
containing CMV enhancer, p-actin promoter, CFTR cDNA, and SV40 late gene polyadenylation. 
The recombinant virus is also deleted of E3 sequences. 
II.B.3. Complementation of the CF defect in vitro 
The function of Ad.CB-CFTR was evaluated in the cell line CFPAC which was derived from the 
pancreatic adenocarcinoma of a CF patient. Infectivity of the cell line with recombinant Ad5 was 
determined initially with lacZ adenovirus and subsequently with Ad.CB-CFTR. CFPAC 
populations exposed to adenovirus at an MOI of 1 demonstrated expression in 30-40% of the 
cells. Recombinant CFTR protein expression was detected by immunocytochemistry in 100% of 
the cells after exposure to Ad.CB-CFTR at an MOI=500 (see Figure 2). The function of 
adenoviral-transduced CFTR protein was assessed by measuring cAMP mediated stimulation of 
Cl conductance using the halide sensitive fluorophone SPQ. In this assay, halide efflux is 
measured by an increase in fluorescence in cells loaded with SPQ and iodide. Results from mock 
infected CFPAC and CFPAC cells infected at an MOI of 500 with Ad.CB-CFTR are shown in Figure 
3. Substitution of NO 3 with iodide in the medium of each population of cells produced little 
change in fluorescence. Subsequent increase in the intracellular cAMP by the addition of 
Recombinant DNA Research, Volume 16 
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