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Ad2/CFTR-2 Gene Transfer Protocol: Single 
Aerosol Administration to the Lung of CF Patients 
’ : 
Studies of Ad2-CFTR-1 
Preclinical Studies of Ad2/CFTR-1. Ad2/l3Gal-1. and Ad2/CMVBGal : Preclinical studies of the related vectors, 
Ad2/CFTR-1 , Ad2/BGal-1 , and Ad2/CMVBGal, include evaluations of the life cycle, safety, and efficacy of the vectors in 
vitro, as well as studies in cotton rats, guinea pigs, and Rhesus monkeys. These studies are encouraging in terms of 
the ability to express functional CFTR and in terms of safety. 
Clinical Study of Ad2-CFTR-1 (Protocol No. CF93-0101) : To evaluate the potential of direct transfer of CFTR cDNA for 
the treatment of CF, Ad2/CFTR-1 was administered to a defined area of nasal airway epithelium of 4 individuals. The 
patients received from 2 x 10 6 III in the first patient up to 5 x 10 7 IU in the last two patients. The last 2 doses represent 
an MOI of 25. The airway epithelium that lines the nasal cavity is similar in morphology and function to the airway 
epithelium that lines the lower respiratory tract. Most importantly for our initial clinical studies, CF causes similar 
abnormalities of electrolyte transport in both the upper and lower airway epithelium. 
In all 4 patients the treatment corrected, focally, the Cl' transport defect that is characteristic of CF-affected epithelia. 
The correction was demonstrated by measurement of the transepithelial electrical potential difference across the nasal 
epithelium which provides an easy and reliable measure of the chloride transport function of the epithelium [15, 16]. 
After treatment, there was a decrease in the elevated basal transepithelial voltage and the normal response to a cAMP 
agonist was achieved. These changes were transient. There was no evidence of viral replication or virus-associated 
adverse effects, even at the highest dose tested. None of the patients exhibited an antibody response to Ad2/CFTR-1 . 
More detailed results of this study were published by Zabner and colleagues [8]. 
The first 2 patients in this study experienced inflammation at the site of Ad2/CFTR-1 administration. This inflammation 
was believed to be related to the application device and/or anesthesia since the same inflammation was observed in 
separate, control CF patients when the identical anesthesia and application procedure was used to apply saline rather 
than Ad2/CFTR-1 . The third and fourth patients did not experience this inflammation when an alternative, non-invasive 
application methodology was utilized to administer Ad2/CFTR-1 in the nasal airway. 
Studies of Ad2/CFTR-2 
Preclinical Studies of Ad2/CFTR-2 In Vitro : The ability of Ad2/CFTR-2 to express CFTR in vitro has been tested in 
human PleLa 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. It has also been shown that Ad2/CFTR-2 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 [17-20] . Such cultures closely resemble the native epithelium, both morphologically 
and functionally. Importantly, the results of the first clinical study in patients with CF using Ad2/CFTR-1 indicate that 
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/CFTR-2, it was found that viral DNA remains episomal. Ad2/CFTR-2 viral DNA 
replication was not detected except in circumstances where El activity is supplied, specifically, when El activity is 
supplied from permissive 293 cells or in coinfections with wild type adenovirus. Moreover, it was found in coinfection 
experiments that wild type Ad2 rapidly outgrows Ad2/E40RF6 + , the parent vector that was used to construct 
Ad2/CFTR-2. These results show that although the Ad2E4'ORF6 + virus is viable in 293 cells, its growth is disabled 
relative to wild-type. 
Preclinical Studies of Ad2/CFTR-2 In Vivo 
Rodent Studies: Cotton rats ( Sigmodon hispidus) are commonly used in assessing safety of recombinant adenovirus 
vectors because they are permissive for viral replication after intranasal inoculation of wild-type adenovirus types 1, 2, 5, 
and 6[21, 22] and also develop pneumonia after treatment, mimicking human adenovirus induced lung disease. In the 
first cotton rat study, the safety of Ad2/CFTR-2 during 3 transtracheal administrations of low (10 8 IU) and high (10 9 IU) 
doses was assessed. Indicators of injury in the lung (BAL protein and total cell numbers) suggested that there was an 
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Recombinant DNA Research, Volume 20 
