Recombinant DNA Advisory Committee - 03/1-2/93 
maximum of 1 x 10 9 pfu per production lot. A maximum of 1 x 10 6 pfu per plate of 
A549 cells were added. This large volume of plates should be manageable. 
With regard to the status of sequencing, Dr. Wilson stated that the vector has been 
sequenced from the LTR through the promoters, through CFTR, through the 
polyadenylation signal, 6.12 kilobases into E2, and 3 kilobases surrounding the E3 
deletion. Hie LTR, CMV, P-actin, the polyadenylation signal, and the CFTR gene were 
intact and functional. However, the sequencing analysis revealed that there are 3 base- 
pair changes. Two of these changes are identical to the rat sequence. This observation 
underscores the importance of sequencing the coding region. 
In an attempt to discover why these base-pair changes occurred, the vector sequence was 
cross-checked with the plasmid and found that these changes were present in the original 
plasmid. Despite these changes, this CFTR allele is totally functional. Currently, the 
investigators are reconstructing the plasmid with known sequences. The new 
recombinant will probably be available in approximately 4 weeks. This protocol will not 
proceed until the sequence of the coding region has been determined. 
Dr. Miller asked how the investigators can be certain that the entire viral DNA sequence 
is acceptable. Dr. Wilson responded that the sequencing is being performed by a 
contractor in 6 different orientations. The sequencing gel has been reviewed, and the 
data are correct. It is unusual that 3 mutations would occur, 2 of which are homologous 
to another species. Dr. Miller asked if the sequence obtained by the contractor has been 
cross-checked with the published sequence. Dr. Wilson said that he learned about the 
base changes only recently and has not had the opportunity to cross-check with the 
published sequence. 
Dr. Parkman said that the standard should be the sequence of the vector that is going to 
be administered to the patient. Dr. Miller explained that the retroviruses have never 
been sequenced, only the plasmids. Even at that, investigators generally assemble 
sequences from the published literature as opposed to sequencing the plasmids 
themselves. The data that Dr. Wilson has provided goes beyond any sequence data that 
has been required previously by the RAC. 
Dr. Wilson explained that the investigators have monitored the presence of antibodies to 
E2a in a variety of species, including primates, and none have been detected. With 
regard to the issue of recombination, Dr. Wilson stated that even in the worst case 
scenario, reconstitution of an E3 deleted wild-type virus should not result in any serious 
adverse consequences. In response to the question about carcinogenesis, Dr. Wilson 
stated that although there are data which demonstrates tumorigenicity in nude mice with 
other serotypes, there is no evidence that type 5 adenoviruses are tumorigenic. 
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Recombinant DNA Research, Volume 17 
