possible to use ex vivo strategies to remove the epithelium, insert the 
normal cDNA and replace the existing epithelium. The adult human airways 
have a surface area of 1-2 m 2 . There are at least 6 major epithelial cell 
types, with the majority of the cells terminally differentiated. Human 
airway epithelial cells can be cultured, but the methods are primitive, the 
differentiated state of the cells is not necessarily the same as that in 
vivo . the growth factors are not known, the normal cell ontogeny is not 
clearly defined, nor is the airway epithelial stem cell population (Rennard 
et al., 1991). Most importantly, the dichotomous branching nature of the 
airways precludes any strategies to remove the epithelium and/or introduce 
corrected autologous airway epithelial cells. Together, these facts argue 
strongly for an in vivo approach to gene therapy. The anatomy dictates that 
this is feasible only through the air side of the epithelium. Unlike the 
lower respiratory tract which gets its blood supply from the pulmonary 
capillaries, the airways are supplied by the bronchial circulation, an 
arterial system comprised of multiple branches derived from the aorta 
(Deffebach and Widdicombe , 1991). Although it is feasible to place cathe- 
ters into the bronchial circulation, their multiplicity and variability 
make this approach very cumbersome. Further, even if the transfer vector 
could be delivered to the airways via the bronchial circulation, the cDNA 
would have to cross the endothelium, the endothelial basement membrane, the 
interstitial space, and the epithelial basement membrane before entering 
the basolateral surface of the epithelium, a very unlikely possibility. 
1.6 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene 
and Mutations 
The gene responsible for CF, the CFTR gene, is a 27 exon gene spanning over 
250 kb on the long arm of human chromosome 7 at q31-q32 (Riordan et al., 
1989; Rommens et al. , 1989; Zielenski et al., 1991). The encoded mRNA is 
about 6500 nucleotides in length. The sizes of the 27 exons vary widely, 
with exon 14b the smallest (38 bp) and exon 13 the largest (724 bp) (Figure 
1.6 -A). Sequence analysis of approximately 10% of the entire CFTR gene has 
shown that all intron/exon junction sequences obey the GT-AG consensus 
rule. The gene includes a number of repetitive elements in introns , includ- 
ing 5 Alu repeats and 1 Kpn repeat and several simple repeats (microsatel- 
lites) , such as (GT) 17 , (GT) 12 , (GATT) 7 , and (TA) U . 
The structure of the putative CFTR gene product is a 1480 residue glycopro- 
tein. There is (N- to C-terminal) a membrane -spanning domain with six mem- 
brane-spanning segments, a nucleotide (ATP) -binding fold (NBF) , a large 
polar R (regulatory) domain which contains multiple potential phosphoryla- 
tion sites, a second similar membrane -spanning domain and a second NBF 
(Figures 1.6-A, 1.6-B) (Riordan et al. , 1989). 
Approximately 220 sequence variations of the CFTR gene have been identi- 
fied, of which about 170 are associated with the clinical manifestations of 
CF. The mutations include missense mutations, nonsense mutations, frame - 
shift mutations, splicing mutations, and small deletions and insertions 
(Collins, 1992). Most of these mutations are scattered throughout the 
coding region of the gene. Many different mutations have been found at the 
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