Recombinant DNA Advisory Committee - 03/1-2/93 
young age. Data has demonstrated that 100% of the population is exposed to adenovirus 
types 1, 2, 5, and 6 by the age of 3 years. This early exposure occurs at a time when the 
lungs are still relatively normal in CF patients. The protocol is designed to treat CF 
patients whose pulmonary function in no longer normal. Dr. Miller explained that 
advanced CF patients do not demonstrate clinical symptoms in response to adenovirus 
exposure. Dr. Straus acknowledged that these patients do not demonstrate bronchiolitis 
or pneumonia. However, these patients are being exposed to low titers and do 
demonstrate evidence of mucosal immunity. These patients have never had large 
quantities of adenovirus particles delivered into their respiratory tract. The capacity of 
these patients is still unknown; therefore, the RAC should not prematurely approve 
excessive levels of wild-type exposure. 
Dr. Parkman said that the criteria for this protocol are similar to those standards 
established for the other CF protocols approved by the RAC. Dr. Miller said that the 
RAC should exercise consistency and ask investigators to demonstrate the ability to 
detect 1 wild-type particle per patient dose. Dr. Post said that there is reason to believe 
that this requirement is unnecessary. Dr. Miller suggested that he would recommend 
approval of the protocol with the stipulation that the investigators try to increase the 
level of sensitivity of their assay for the detection of wild-type adenovirus; however, the 
current standards are acceptable at this time. 
Dr. Haselkorn suggested that perhaps the level of PCR sensitivity could be increased by 
the addition of biotinylated nucleotides in the late steps and using radiolabelled avidin to 
detect the product. Another option is to perform a reconstruction experiment with 1 x 
10 12 vector particles and identifying replication-competent virus following serial passage. 
Dr. Trapnell explained that one of the difficulties in identifying replication-competent 
particles is the number of cells that must be inoculated. This procedure could require as 
many as 2 x 10 4 plates to perform 1 assay. Even if this number of plates could 
technically be inoculated, it would be difficult to believe 1 plaque in 2 x 10 4 plates. 
Dr. Trapnell said that it is reasonable to believe that the level of PCR sensitivity will be 
improved. However, even in the worst case scenario, these patients would not be 
exposed to levels of wild-type virus that are any higher than the natural situation. Dr. 
Post asked if assays have been performed with A549 cells. Dr. Trapnell said that assays 
have been performed with A549, HeLa, human embryonic kidney, and 293 cells. There 
are problems associated with each of these cells. These systems reach their capacity in 
terms of the number of vector particles per cell; therefore, dilutions are still necessary. 
Dr. Parkman reminded the RAC that the limit of 1 x 10 3 is the worst case scenario of 
replication-competent particles; the actual number could be 0. Dr. Trapnell agreed with 
Dr. Parkman noting that the initial group of patients who will receive 1 x 10 9 vector 
particles will receive vector preparations that have been demonstrated to have less than 
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Recombinant DNA Research, Volume 17 
