V.B.5. Previous Experience Using Adenovirus in Human Clinical Trials 
for the Treatment of Cystic Fibrosis 
Dr. James M. Wilson and colleagues at the University of Pennsylvania have initiated a gene therapy 
phase I clinical to test the safety and toxicity of recombinant adenoviruses for the potential use in 
cystic fibrosis patients. The original protocol was submitted to the Recombinant DNA Advisory 
Committee (R.A.C.) in the Fall of 1992 and approved soon afterwards (see Wilson, 1994). 
The basic protocol is as follows. Patients were taken to the bronchoscopy suite where baseline 
bronchoalveolar lavage (BAL) and bronchial brushing samples are obtained from a segment in the 
left lower lobe. Four days later, the patient returned to the bronchoscopy suite where BAL and 
bronchial brushings were obtained from the segment that received the virus. Approximately seven 
days after initial virus instillation, nasopharyngeal and rectal swabs were analyzed for the presence 
of viral DNA using a sensitive PCR test. When the patient demonstrated negative PCR analysis of 
three serial rectal and nasopharyngeal samples, he or she was discharged from the hospital. 
Samples recovered from BAL and bronchial brushings were analyzed for evidence of recombinant 
gene expression using a variety of molecular, biochemical and functional assays. 
The first seven patients were adults, ranging from 22 to 36 years of age, with equal distribution 
between males and females. To date there have been no adverse clinical outcomes, either as a 
consequence of the bronchoscopies or in response to the virus. No significant findings have been 
observed in these studies except for the transient (lasting less than six hours) development of an 
infiltrate in the area of the lung that was lavaged. This was likely due to retained fluid that was 
quickly absorbed. One very consistent finding was a transient elevation in liver transaminases at 
the beginning of the protocol found in most of the patients. This was not associated with clinical 
evidence of hepatitis and the biochemical profile did not suggest an obstructive picture. Serum 
transaminases declined to normal levels within one week after gene transfer. 
Careful assessment of the patients for shedding of recombinant virus was performed. It was 
necessary to demonstrate the absence of virus in nasopharyngeal and rectal samples on three 
consecutive days following instillation of virus before discharge was authorized. The specific assay 
that was used is based on PCR amplification of the viral genome with transgene specific 
oligonucleotides. Samples were also analyzed for recovery of Ad5 sequences using a similar 
approach with oligos spanning the hexon gene. Vigorous swabbing of nasopharyngeal and rectal 
mucosa allowed recovery of enough biological material for extraction of DNA. The purified, 
recovered DNA was subjected to PCR amplification using nested primers. This analysis allowed 
detection of 1*10 viral genomes per tube. These analyses failed to detect viral DNA in samples 
recovered after virus administration from patients CF2 through CF7. Initial PCR analysis of 
nasopharyngeal swabs of patients CF1 were positive on days 1 and 3 following instillation of virus. 
Subsequent studies indicated that this was likely due to a false positive reading and the patient 
subsequently remained negative. 
In order to detect and quantitate gene expression in airway epithelial cells, inflammatory cells in 
BAL, as well as epithelial cells isolated by bronchial brushing of the distal and proximal portion of 
the bronchus that received virus were recovered. Samples obtained on the contralateral side prior 
to virus instillation were used as controls. The molecular characterization of these samples using 
techniques of PCR for DNA and RNA as well as in situ hybridization were performed in the Cell 
Morphology Core. Hybridization to the antisense probe gave a nonfocal diffuse background in cells of 
proximal and distal airway that were harvested four days after gene transfer. This signal was 
indistinguishable from that obtained with the sense probe. This negative result indicates little, if 
any, gene transfer obtained with this dose of virus. 
Another scientific question addressed in this protocol was an evaluation of the recipient's response to 
the recombinant virus. Peripheral blood mononuclear cells are being harvested and cryopreserved 
for subsequent analysis of CTL and helper T cell function. Sera was also analyzed for neutralizing 
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Recombinant DNA Research, Volume 20 
