Recombinant DNA Advisory Committee - 03/1-2/93 
method is to evaluate the biologic titer of infected cells. The second method is to look 
for cytopathic effects and to evaluate for characteristic cell morphology which would 
indicate wild-type infection. The third method, which is more sensitive than the other 2 
methods, is metabolic labeling of DNA in infected cells. The metabolic labeling method 
identifies the presence of wild-type genomic sequences, i.e., El sequences. 
Dr. Trapnell presented data demonstrating metabolic labeling of genomic DNA 
sequences in bronchial epithelial cells that were obtained by bronchoscopy. Following 
DNA extraction, enzyme restriction analysis is performed using agarose gel 
electrophoreses and autoradiography. The wild-type and recombinant virus have 
distinguishable restriction fragment patterns. PCR analysis is being used to detect the 
presence of replication-competent adenovirus particles. The PCR method is several logs 
more sensitive than the other methods. A reconstruction experiment was presented in 
which wild-type virus was mixed with the recombinant virus at different ratios. The cells 
are heat denatured, and PCR amplification is performed with both the Ela and E2b 
primers. Next, these samples are subjected to Southern blot analysis. The PCR 
amplification method demonstrates that 1 wild-type virus particle can be detected in 1 x 
10 9 vector particles. This assay sensitivity is the current level. 
Dr. Miller said that patients will receive as much as 1,000 times the number of vector 
particles used in the reconstruction experiment. Dr. Parkman said that with this level of 
sensitivity, the investigators can only say that there is less than 1,000 replication- 
competent particles per patient dose. Dr. Trapnell said that plaque purification will be 
performed; therefore, the theoretical number of replication-competent particles will be 
much less than 1,000. Ms. Grossman inquired if there are other cell-based assays that 
could be performed to increase the level of sensitivity. Dr. Trapnell responded that the 
cytopathic effect assay could be performed; however, the necessary dilutions would 
require thousands of plates, which would make assay prohibitive. 
Dr. Trapnell presented data from other trials in which wild-type adenovirus was 
delivered to the respiratory tract of a large number of individuals. This trial was 
conducted between 1953 and 1970. These individuals received as much as 4 x 10 5 wild- 
type particles. The most severe symptoms consisted of mild upper respiratory illness, i.e., 
coughing and rhinorrhea. These symptoms were consistent with those observed in 
patients with a typical adenovirus induced cold. For individuals who demonstrated 
preexisting neutralizing antibody from a prior infection, there was an inverse relationship 
between the amount of antibody and the development of asymptomatic infections. 
Patients enrolled in this study will have demonstrated neutralizing adenovirus antibody. 
There has never been any instance of malignancy associated with adenovirus infection in 
humans. 
Dr. Motulsky inquired if these trials have been followed long-term. Dr. Trapnell said 
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Recombinant DNA Research, Volume 17 
