3.1.2 Expression of CFTR Protein 
Following infection with AdCFTR (but not in uninfected cells), human CFTR 
protein can be detected by immunohistochemistry in a variety of cell lines 
and freshly isolated cells, including 293 cells, HeLa cells, IB3-1 cells, 
freshly isolated normal human bronchial cells, freshly isolated cystic 
fibrosis human bronchial epithelial cells, freshly isolated cotton rat 
tracheal-bronchial cells, and freshly isolated rhesus bronchial cells (see 
Appendix 1, Figure 3.1.2-A, Figure 3.1.2-B). Using identical methods, human 
CFTR protein can also be detected following AvlCFl infection of 293 cells, 
HeLa cells, IB3-1 cells, and freshly isolated normal human bronchial 
epithelial cells (not shown). In contrast, expression of the endogenous 
CFTR gene in these cells is low and endogenous human CFTR protein cannot be 
detected in freshly isolated tissues by this immunohistochemistry assay. 
This is true for cells from experimental animals and from humans. 
Following infection with Ad-CFTR (but not control vectors, or in uninfected 
cells) , human CFTR protein can be immunoprecipitated and subsequently 
detected after phosphorylation with protein kinase A from a variety of 
cells including 293, freshly isolated normal human airway epithelial cells, 
and freshly isolated cotton rat tracheal -bronchial epithelial cells (Figure 
3.1.2-C). 
Following infection with Ad-CFTR (but not control vectors, or in uninfected 
cells), de novo synthesized human CFTR protein can be detected by 35 S- 
methionine labeling, immunoprecepitation , sodium dodecyl sulfate 
polyacrylamide gels, and autoradiography in 293, CFPAC-1 and freshly iso- 
lated normal human airway epithelial cells (Figure 3.1.2-D and Appendix 1) . 
3.1.3 Functional "Complementation" of the Cystic Fibrosis Phenotype 
Consistent with the role of the CFTR protein as a cAMP-regulatable Cl - 
channel in epithelia, the biologic phenotype of CF epithelia is the in- 
ability to secrete Cl - in response to an increase in intracellular levels 
of cAMP. This can be corrected by infection of CF epithelial cells with Ad- 
CFTR or AvlCFl. The assay used to demonstrate this utilizes quantification 
of 36 C1 - secretion in response to increased cAMP (see Appendix 1 for de- 
tails) . An example of the functional consequences of AvlCFl infection 
demonstrated with the 36 C1 - assay in epithelial cells from individuals with 
CF (CFPAC-1) is shown in Figure 3.1.3-A. In contrast, a control vector had 
no effect. The vector AdCFTR will convey cAMP stimulatable Cl - secretion to 
cells that do not normally have this function, and to pancreatic cells and 
airway epithelial cells from individuals with cystic fibrosis (Appendix 1, 
Figure 3 . 1 . 3-B) . 
3.1.4 Can the Vector Survive in the Airway Epithelial Lining Fluid of Indi- 
viduals with Cystic Fibrosis? 
The airway epithelial milieu of individuals with CF is characterized by an 
intense neutrophil dominated inflammatory process, and with it, inflamma- 
tory cell derived mediators including oxidants, proteases and a variety of 
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Recombinant DNA Research, Volume 16 
