8.4 Evaluation of Biological Activity 
The principal measures of biological activity will be as follows: 
8.4. 1 DNA in situ PCR on brushed bronchial epithelial cells, utilizing vector specific 
oligonucleotide primers, to evaluate the efficiency of vector DNA transfer. This 
assay will quantitate the percentage of cells transduced and the distribution of vector 
gene transfer. 
8.4.2 RNA-PCR, standard RNA in situ hybridization, RN A in situ PCR, and Northern 
blotting to evaluate vector-produced CFTR RNA expression in brushed nasal and 
bronchial epithelial cells. RNA-PCR will provide a sensitive indicator of vector 
RNA expression, and will be particularly useful in determining the duration of 
vector expression. Northern blotting will provide a more quantitative assessment. 
RNA in situ hybridization and PCR techniques provide the ability to identify which 
cell types within the airway are expressing recombinant CFTR message. 
8.4.3 Western blotting, immunohistochemistry and immunofluorescence to evaluate 
recombinant human CFTR protein expression. Protein assays will include both 
semi-quantitative Western blot for detection of CFTR protein from cell extracts, 
while immunostaining techniques allow identification of the cell types which are 
expressing recombinant CFTR protein. 
8.4.4 Controls for each of these assays will consist of brushed cells from the nose and 
bronchus of the same patients to be used as subjects. Control cells will be obtained 
on days -4 and 0. Experimental samples will be collected on days 10, 30, 60, 
90, 180, 270, and 365. If there is no evidence of CFTR expression in the nose for 
any 2 consecutive time points then the remaining assessments will be altered to omit 
nasal brushing. If there is no evidence of CFTR expression in the lung for any 2 
consecutive time points then the remaining assessments will be altered to omit 
fiberoptic bronchoscopy. All of the above studies are available through the Johns 
Hopkins CF Gene Therapy Center Expression Core and in the principal 
investigator's laboratory. 
8.5 Evaluation of Physiologic Activity 
The principal measures of physiologic activity will be: 
8.5.1 Direct nasal transepithelial potential difference, with baseline measurement taken at 
days -4 and 0, and post-vector instillation readings performed on days 3, 10, 30, 
60, 90, 180, and 365. Each assessment will consist of both baseline readings and 
amiloride responses. The principal investigator and co-investigators have extensive 
experience with nasal PD measurement and will be assisted by the clinical core in 
the performance of these repetitive measurements; 
8.5.2 36q- efflux and SPQ fluorescence of primary cell cultures derived from brushed 
nasal and bronchial specimens before and after vector instillation. Demonstration of 
cAMP-mediated increase in the rate of chloride efflux will be used as an indicator of 
normal CFTR function. The Johns Hopkins Gene Therapy Center tissue culture 
and expression core will assist with these repetitive assays. This approach can 
readily be applied to both nasal and bronchial epithelial cells obtained by brushing; 
8.5.3 Other indicators previously described in the toxicity monitoring section. 
[128] 
Recombinant DNA Research, Volume 20 
