M.J. Welsh and A.E. Smith, RAC Application 
B.2.b.(3) Explain in detail all results from animal and cultured cell model experiments 
which assess the effectiveness of the delive ry system (part 2,a. above) in achieving the 
minimally required level of gene transfer and expression (2.h. ( 2 ) ahovel 
1. Cultured cell models 
a. Studies with Ad2/BGaI-l 
We infected these cells [Point B.2.b.(l)] in tissue culture and assayed for B-galactosidase 
activity. We used B-galactosidase tagged with a nuclear localization signal because 
background blue staining with X-gal is a common problem, particularly with airway cells. 
After exposure to Ad2/BGal- 1 , B-galactosidase activity was detected as a strong nuclear- 
localized blue staining in a variety of the different cells when infected at a multiplicity of 
infection between 0.01 and 30 PFUs per cell. 
b. Studies with Ad2/CFTR-1 
After exposure of these cells to Ad2/CFTR-1, we detected CFTR by immunoprecipitation 
followed by phosphorylation and autoradiography. We also detected CFTR by 
immunocytochemistry. 
Because it is a chloride channel, CFTR can also be detected using assays based on its 
function. We measured cAMP-dependent CFTR chloride channels using the halide- 
sensitive fluorophore 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ). In the SPQ assay, 
an increase in halide permeability through CFTR chloride channels results in a rapid 
increase in SPQ fluorescence (9). We infected CF nasal epithelial cells with Ad2/CFTR-1 
at a multiplicity of 5 PFU/cell. Stimulation of Ad2/CFTR-1 -infected cells with forskolin 
(20 |iM) and IB MX (100 |iM) increased SPQ fluorescence. In contrast, cAMP did not 
increase halide permeability in uninfected or wild-type adenovirus-infected (5 MOI) cells. 
Thus, only cells infected with Ad2/CFTR-1 expressed c AMP-regulated CFTR chloride 
channels. Some Ad-CFTR-infected cells also showed an increased basal halide 
permeability that was independent of cAMP stimulation. Although we have not studied 
this in detail, one interpretation of these data is that the adenovirus-infected cells have an 
elevated endogenous level of cAMP. We have obtained similar results with 293 cells. In 
human airway epithelial cells, we found that 15-30% of cells expressed CFTR as assessed 
by the acquisition of cAMP-dependent stimulation of halide permeability. 
These results indicate that the adenovirus vector can direct the expression of functional 
CFTR chloride channels and B-galactosidase in a variety of cultured epithelial cells. 
2. Primary cultures of human airway epithelia grown on permeable supports. 
a. Studies with Ad2/BGal-l 
To test vector efficacy, we examined the effect of Ad2/BGal-l added at a variety of 
multiplicity's of infection (MOI) on primary cultures of normal human airway epithelia 
grown on permeable filter supports (Fig. 9). As expected, the number of stained cells 
depended upon the MOI; blue staining of a small percentage could be detected at an MOI 
as low as 0.01 PFU/cell. As the multiplicity of infection increased, so did the number of 
cells with blue-staining nuclei. In some cases, cells were treated with neuraminidase (0.2 
U/ml) and dithiothreitol (5 mM) to remove mucus and cell debris prior to addition of virus; 
however, this treatment did not appear to increase the percentage of stained cells. Blue- 
staining cells persisted for up to 20 days after infection with Ad2/BGal-l at 5 MOI. Longer 
studies were limited by the short "lifetime" of the epithelial cultures. 
Recombinant DNA Research, Volume 16 
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