Critical to our understanding of the pathophysiology of CF lung disease is a 
precise definition of the cellular and subcellular localization of CFTR in the human 
airway. Previous studies have reported variable success in accomplishing this by 
direct localization. Immunocytochemical analyses of adult human tissues have 
successfully localized CFTR in epithelial cells of sweat ducts, small pancreatic ducts, 
kidney tubules and intestinal crypts, but failed to detect CFTR in the lung (10,11). 
Analysis of RNA extracted from human bronchial brushings of normal patients 
indicated that the CF gene is expressed at low levels in the cells released by this 
procedure, which were primarily ciliated and nonciliated columnar cells of the 
surface epithelium (31). In situ hybridization studies of rat trachea show low levels 
of diffuse staining over the surface epithelium but these studies could make no 
conclusions about the presence of CFTR mRNA in submucosal glands which are 
extremely nonabundant in the rodent airway. More recently, Zeitlin et al. detected 
CFTR protein in primary nasal epithelial cells by Western blot analysis (32). 
Function of CFTR has been clearly demonstrated in cells isolated from the surface 
epithelium using electrophysiological techniques [Reviewed in (14)]. 
Techniques of in situ hybridization and immunocytochemistry were used to 
characterize the distribution of CFTR expression in bronchus from non-CF and CF 
tissues (33) (see Appendix A). 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, AF508, is associated with markedly decreased levels of 
protein in all structures, and the less common mutation, G551D allele, is associated 
with normal distribution of apically localized protein. 
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 surface epithelia, is based on the premise that correction of 
the submucosa is not necessary for some level of efficacy. 
II.B.3. Strategies for Gene Therapy of CF Lung Disease 
Two general approaches to correction can be envisioned: the so-called 
transient, or nonintegrative, expression of the CFTR cDNA; and integrative 
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