M.J. Welsh and A.E. Smith, RAC Application 
Ad2-ORF6/PGK-CFTR 
Item 5 - Clinical Protocol 
Studies of Ad2-ORF6/PGK-CFTR In Vivo. 
We have used cotton rats ( Sigmodin hispidus ) for several studies because they have been shown to be 
permissive for viral replication after intranasal inoculation of wild-type adenovirus types 1, 2, 5, and 6 
(36,37). Treated cotton rats also develop pneumonia and thus can mimic human pathology induced by 
adenovirus. In Appendix 7.4, we describe studies in which we have investigated the efficacy of 
adenovirus vectors and the safety of Ad2-ORF6/PGK-CFTR during repeated administration. Although 
we observed some variability in expression, we observed no difference in expression of recombinant 
protein despite 4 repeated administrations of an adenoviral vector. More importantly, although we 
observed occasional patchy inflammation as indicated by a macrophage infiltrate, it did not correlate with 
treatment, i.e.., it was the same in treated and control animals. 
We studied Rhesus monkeys (. Macaca mulatto) because the physiology and anatomy of the airway 
epithelium closely resembles that of humans (38). For our proposed studies in humans, we plan to use 
the upper airway epithelium. Therefore, we tested the safety and efficacy of repeat administration of 
adenovirus vectors in the nasal epithelium. We applied Ad2/CFTR-1 on three occasions (results reported 
in Appendix 7.6). Then we applied Ad2-ORF6/PGK-CFTR to the nasal epithelium on two separate 
occasions for a total of 5 administrations. Although all three monkeys had positive antibody titers to 
adenovirus, none of the monkeys developed any clinical signs of viral infection or inflammation, either 
systemically or in the nasal epithelium. There was no viral replication and biopsies and brushings of 
treated epithelium showed no evidence of inflammation. Repeated treatment did not prevent expression of 
CFTR. Results of in vivo studies are described in detail in Appendix 7.4 
SPECIFIC AIMS 
The foregoing considerations indicate that gene therapy would be a major advance in the treatment of CF. 
They also suggest that adenovirus may be a good vector system for transfer of CFTR cDNA to correct the 
CF pulmonary defect. However, use of adenovirus vectors as a treatment for CF lung disease will 
require repeat administration and administration at higher doses than we have previously used in our 
initial study in humans. Moreover, the use of and further development of adenoviral vectors will require 
some demonstration of clinical efficacy in CF airway epithelia involved by the disease. Therefore, in this 
application we propose to address these issues. 
Part A. Safety of Dose and Repeat Administration in the Nasal Epithelium. 
1. Is repeat administration of an adenoviral vector safe? We will examine the effect of repeat 
administration of Ad2-ORF6/PGK-CFTR to the nasal epithelium to determine if it causes an inflammatory 
or immune response. We will also determine if the efficacy of Ad2-ORF6/PGK-CFTR in correcting the 
CF chloride transport defect decreases with repeated administration. 
2. What is the effect of vector dose on safety and efficacy? We will examine the effect of 
progressively increasing doses of Ad2-ORF6/PGK-CFTR on safety and biochemical efficacy. 
Part B. Clinical Efficacy in the Maxillary Sinus. 
1. Does adenovirus-directed CFTR expression have clinical efficacy? We will test the 
clinical efficacy of Ad2-ORF6/PGK-CFTR in correcting the airway epithelial disease observed in the 
maxillary sinus. We will examine the effect of repeat administration, use one maxillary sinus as a control, 
and assess clinical efficacy using computed tomography scans as well as cellular and biochemical 
measures of efficacy. The data on clinical efficacy will also assess the balance between efficacy in 
correcting the biochemical defect and any potential adverse effects associated with vector administration. 
2. Is repeat administration of an adenoviral vector safe? We will examine the effect of repeat 
administration of Ad2-ORF6/PGK-CFTR to determine if it causes an inflammatory or immune response. 
Recombinant DNA Research, Volume 18 
[867] 
