accomplished in a non-replicating, stable target cell and the recombinant gene 
should be expressed for a prolonged period of time without pathology. In a series of 
studies performed in Cotton rats, Crystal and colleagues have provided convincing 
evidence that recombinant adenoviruses may have the properties described above 
necessary for safe and effective gene transfer to the human airway (38,39). 
Adenoviruses are non-enveloped DNA viruses responsible for many types of 
self-limited respiratory illnesses in humans (40,41). Many serotypes of adenoviruses 
exist, some of which have been exposed to a large proportion of the population in 
the United States. 
Adenoviruses have been the subject of extensive investigation in the 
laboratory (42-44). This has proven to be a powerful system for studying basic 
mechanisms of gene regulation. The adenovirus exists as a 35 kb double stranded 
DNA molecule with multiple early and late transcriptional units. The type of 
recombinant virus proposed in this protocol has been used as a vector system in 
many in vitro and in vivo settings (45). The El region is deleted so as to render the 
virus defective in replication. Under some conditions, such as high Multiplicity of 
Infection (MOI), El deleted viruses can replicate in recipient cells. The 
recombinant minigene is placed under the control of a heterologous promoter in 
place of El sequences. 
Perricaudet and colleagues have developed El deleted Ad5 for use in 
vaccines (46). They have performed experiments in which hepatitis B surface 
antigen expressing virus was injected into nonhuman primates. Interestingly, no 
immune response was observed. Of relevance to this protocol was the study by 
Smith et al. in 1970 in which volunteers were exposed to type 4 adenovirus by 
inhalation as part of a vaccination trial (47). No substantial morbidity was noted. 
II.B.4. Efficiency of gene transfer required for therapy of CF lung disease 
CFTR functions as a cAMP-regulated Cl" channel. The number of 
functional Cl" channels on a per-cell basis required to account for the rates of 
airway transepithelial Cl" secretion is very low (20-100 channels per cell). Thus, the 
number of functional CFTR molecules required per cell for full physiologic activity 
of the cell is small. These functional data are congruent with data reported on the 
level of CFTR expressed per cell on the basis of analyses of mRNA and/or protein. 
Studies of in situ hybridization reported above from our own laboratories observed 
that the level of expression of CFTR transcripts in the superficial epithelial cells was 
very low [(33); see Appendix A]. These data are complemented by the studies of 
Crystal and co-workers, who, using PCR-based techniques, have also found that the 
number of CFTR transcripts per cell is extremely low [as low as one to two 
transcripts per cell (31)]. Finally, these data are complemented by studies of 
distribution of CFTR protein in airway epithelial cell lines. In studies in which 
CFTR was recognized by Western blot analysis, large numbers of cells (more than 
10^) were required to produce sufficient plasma membrane mass to recognize 
CFTR by immunologic techniques (48). Taken together, these data suggest that 
relatively little CFTR per cell must be introduced to effect restoration of normal Cl" 
transport in CF cells. 
Recombinant DNA Research, Volume 17 
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