M.J. Welsh and A.E. Smith, RAC Application 
Ad2-ORF6/PGK-CFTR 
Item 5 - Clinical Protocol 
similar, b) Assessment of CF clinical disease is easier and entails less risk than equivalent assessment of 
lung disease, c) Because there is no physical connection between the two maxillary sinuses, we can use 
one sinus as a control, d) The dose of virus is relatively small because of the limited area of application, 
e) The risk to participants is minimized if an adverse reaction should occur in the sinuses as compared to 
the risk with lung administration. 
DESIGN OF Ad2-ORF6/PGK-CFTR. 
The vector to be utilized in the present protocol was derived from Ad2 DNA and CFTR cDNA. It is 
named Ad2-ORF6/PGK-CFTR. The DNA construct comprises a full length copy of the Ad2 genome of 
approximately 37.5 kb, from which the early region 1 genes (present at the 5' end of the viral DNA) have 
been deleted and replaced by an expression cassette encoding CFTR. The expression cassette includes 
the promoter for phosphoglycerate kinase (PGK) and a poly A addition site from the bovine growth 
hormone gene. In addition, the E4 region of Ad2 has been deleted and in its place the open reading frame 
6 of the Ad2 E4 region has been inserted. Appendix 7.1 describes in detail the design and construction of 
the Ad2-ORF6/PGK-CFTR vector. 
STUDIES OF Ad2-ORF6/PGK-CFTR. 
Studies of Ad2/CFTR-1, Ad2/BGaI-l, and Ad2/CMV-BGal. 
Many of our studies of the related vectors - Ad2/CFTR-1, Ad2/BGal-l, and Ad2/CMV-BGal - are 
described in Appendices 1. 5-1.1 and our previous RAC proposal. The experiments include studies of the 
life cycle, safety, and efficacy of the vectors in vitro , as well studies in cotton rats, guinea pigs, and 
Rhesus monkeys. Appendix 7.8 describes the results of our first study using Ad2/CFTR-1 in humans. 
These studies are encouraging in terms of the ability to express and in terms of safety. 
Studies of Ad2-ORF6/PGK-CFTR In Vitro. 
We have tested the ability of Ad2-ORF6/PGK-CFTR to express CFTR in several cell lines, including 
human HeLa cells, human 293 cells, and primary cultures of normal and CF human airway epithelia. 
When these cells were grown on culture dishes, the vector was able to transfer CFTR cDNA and express 
CFTR as assessed by immunoprecipitation and by functional assays of halide efflux. We have also 
shown that Ad2-ORF6/PGK-CFTR can correct the cAMP-stimulated chloride secretion defect in primary 
cultures of CF airway epithelial cells grown on permeable filter supports at the air-liquid interface (32- 
35). Such cultures closely resemble the native epithelium, both morphologically and functionally. 
Importantly, the results of our first study in humans with CF (Appendix 7.8), indicate that our data 
obtained with primary cultures of human airway epithelia grown on permeable filter supports predict the 
results in the airway epithelium of patients. 
In studies of the life cycle of Ad2-ORF6/PGK-CFTR, we found that viral DNA remains episomal. There 
was no evidence of viral DNA synthesis and no evidence of DNA instability. We have been unable to 
detect viral replication except in the permissive 293 cells. The functions of El could, however, be 
complemented by superinfection with a wild-type virus. To examine viral DNA synthesis in doubly 
infected 293 cells we took samples at different times following infection with wild-type and the Ad2E4' 
ORF6+ virus and examined adenovirus DNA following restriction enzyme digestion. We found that 
wild-type DNA can be detected by the appearance of characteristic DNA fragments and that these 
fragments rapidly overgrow the Ad2E4'ORF6 + backbone virus. This result implies that although the 
Ad2E4'ORF6 + virus is viable in 293 cells, its growth is disabled relative to wild-type. This is perhaps 
not surprising since it lacks E40RF6/7 and ORF3. These results argue that Ad2-ORF6/PGK-CFTR may 
be disabled relative to wild-type in the event of an inadvertent subsequent infection of a treated patient 
with wild-type virus. 
We have examined the preparations of Ad2-ORF6/PGK-CFTR by the HeLa cell assay and by the PCR 
reaction described above. We have not detected any wild-type recombinant virus by these assays. Thus, 
at the sensitivity levels of these assays we have not detected any recombination events. We are currently 
refining these assays to enable us to demonstrate the absence of wild-type adenovirus in the highest doses 
proposed here. Results of in vitro studies are described in detail in Appendix 7.2-7. 3. 
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Recombinant DNA Research, Volume 18 
