3. Experimental Evidence Supporting the Use of a Replication Deficient 
Recombinant Adenovirus Containing the Human CFTR cDNA to Treat the 
Respiratory Manifestations of Cystic Fibrosis. 
There is evidence from a variety of viewpoints that a El - E3“ replication 
deficient recombinant adenovirus can transfer the normal human CFTR cDNA to 
airway epithelial cells and that the CFTR cDNA is expressed and will cor- 
rect the CF phenotype of epithelial cells from individuals with CF. For 
additional details, see Appendix 1 (Rosenfeld MA, Yoshimura K, Trapnell BC, 
Yoneyaraa K, Rosenthal ER, Dalemans W, Fukayama M, Bargon J, Stier LE, 
Stratford-Perricaudet L, Perricaudet M, Guggino WB , Pavirani A, Lecocq J-P, 
Crystal RG. In vivo transfer of the human cystic fibrosis transmembrane 
conductance regulator gene to the airway epithelium. Cell 1992; 68:143-155) 
and Appendix 2 [Mastrangeli A, Danel C, Rosenfeld MA, Stratford-Perricaudet 
L, Perricaudet M, Pavirani A, Lecocq J-P, Crystal RG . Diversity of airway 
epithelial cell targets for in vivo recombinant adenovirus -mediated gene 
transfer. J Clin Invest (in press)]. 
3.1 In vitro Evidence 
The in vitro evidence demonstrates that a recombinant adenovirus vector 
containing the CFTR cDNA will transfer the cDNA in a fashion that results 
in expression of the CFTR gene at the mRNA and protein levels and will 
complement the abnormal mutations in the endogenous CFTR genes to reestab- 
lish the ability of the epithelial cells to secrete Cl~ in response to 
cAMP. Further, such a vector will survive in the relatively harsh inflamma- 
tory milieu of the airway epithelial lining fluid in CF. 
3.1.1 Expression of CFTR mRNA 
Following AdCFTR or AvlCFl infection, human CFTR mRNA derived from the 
adenovirus vector can be detected in a variety of cell lines including 
human cervical carcinoma (HeLa) , cystic fibrosis pancreatic carcinoma cell 
line (CFPAC-1), human embryonic kidney cells (293), and freshly isolated 
cystic fibrosis human bronchial cells (data for AdCFTR, see Appendix 1; 
data for AvlCFl, not shown). Following infection with AdCFTR (but not with 
controls) , in situ hybridization demonstrated human CFTR mRNA in freshly 
isolated normal human bronchial epithelial cells and cystic fibrosis human 
bronchial epithelial cells (see Figures 3.1.1-A, 3.1.1-B). 
Following infection with AdCFTR, polymerase chain reaction (PCR) evaluation 
of freshly isolated human bronchial cells derived from individuals with 
cystic fibrosis demonstrated AdCFTR-directed mRNA transcripts (Figure 
3.1.1-C). Further, whereas prior to infection and after infection with a 
control virus, these cells expressed only mRNA transcripts with the endoge- 
nous CFTR transcripts expressing the CF mutations. After AdCFTR infection, 
normal CFTR transcripts were present, as demonstrated by sequence analysis 
and by hybridization with specific probes (not shown) . 
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