pulmonary epithelial cells does not alter lung growth, development or function making feasible 
the efforts to transfer the CFTR cDNA in a widespread manner. 
1.8.1 Pulmonary Targets for Correction of Cystic Fibrosis by Gene Therapy: While cystic 
fiblrosis involves pathology in a variety of organs, most of the morbidity and early death 
associated with cystic fibrosis relates to abnormalities of the respiratory tract. The CFTR 
protein is expressed in epithelial cells in a variety of tissues; however, the epithelial cells of the 
lung provide the major focus for gene therapy to correct the pulmonary abnormalities associated 
with the disease. The CFTR mRNA is expressed at relatively low levels in most organs and the 
distribution of the CFTR protein has not been readily discemable in most tissues by standard 
immunocytochemical techniques. CFTR mRNA has been detected in lung and other organs by 
a variety of methods including reverse transcription-PCR (appendix 1, Trapnell et.al., 1991a), 
S, protection (see section 3 of this proposal) and by northern blot hybridization analysis (Riordan 
et.al., 1989, Tresize et.al., 1991, Fiedler et.al., 1991, Whitsett et.al., 1992). We have recently 
demonstrated that the CFTR mRNA in mouse lung is confined to epithelial cells of the 
respiratory tract (Whitsett et.al., 1992). The abundance of the CFTR mRNA is regulated in a 
developmental manner, being highest in fetal lung (Figure 1), decreasing dramatically prior to 
birth, and approaching adult levels in the first postnatal weeks (Wert et.al., 1992). In the adult 
murine lung, CFTR mRNA was found to be distributed relatively homogeneously throughout 
the respiratory epithelium and was not confined to specific cell types (Figure 1). In the whole 
mount in situ hybridization analysis of the fetal mouse (day 13-15), the CFTR mRNA was 
observed only in epithelial cells and was most abundant in testes, intestine, salivary duct and in 
the lung. In the human lung, CFTR mRNA has been detected in lung epithelial cells of the mid- 
trimester human fetus by in situ hybridization analysis (McCray 1992). Low abundance of the 
CFTR mRNA was detected by reverse transcription-pcr of the RNA from human airway cells 
obtained by brushing the tracheal-bronchial epithelium during bronchoscopy (Trapnell 1991a). 
However, in all analyses to date, CFTR protein has not been readily detectable by available 
antibodies in pulmonary tissues. Present data supports the concept that the hCFTR mRNA is 
present at relatively low levels throughout the respiratory epithelium, including both distal and 
proximal regions of the lung. The percentage of cells that need to be corrected to ameliorate 
lung disease is also not known at present. However, recent work by Johnson et.al., (1992) 
demonstrated that ion conductance across CF epithelial cell monolayers could be corrected by 
expression of CFTR mRNA in only 6-10% of the cells comprising the monolayer. While it 
remains unclear whether correction of CF in vivo will require precise temporal-spatial regulation 
of CFTR expression in the lung, the data at present, demonstrate the presence of CFTR mRNA 
in virtually all epithelial cell types of the respiratory tract, providing a broad target for 
expression of the transferred CFTR gene. Since adenovirus is efficient and able to infect a wide 
variety of pulmonary epithelial cells in vivo and in vitro , this vector appears reasonably suitable 
for transfer of the CFTR gene to the respiratory tract. 
1.8.2 Strategies for Gene Therapy of the Respiratory Epithelium: Genes may be transferred 
by a variety of methodologies in vitro , however, only a few strategies may be applicable for 
gene transfer to the respiratory epithelium in vivo. At present, these include the use of viral 
vectors including retroviruses, adenoviruses, adeno-associated viruses, plasmids, liposomes or 
DNA-carrier complexes. Receptor systems endogenous to the respiratory tract or viral proteins 
which might aid in the delivery and integration of the DNA into the respiratory tract are also 
being developed. The respiratory epithelium undergoes very slow regeneration (estimated half 
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