Recombinant DNA Advisory Committee - 12/3-4/92 
in cell distribution ranging from neutrophils to macrophages. Transient expression of 
both lac/Z and CFTR were observed on day 3 in the segments in which they were 
introduced. Studies have been initiated in the cotton rat model to evaluate toxicity and 
distribution of gene expression. 
Revisions have been made to the clinical protocol regarding: (1) changes in vector 
dosages, and (2) elimination of the transbronchial biopsy procedure. Patients 1 through 
3 will receive 10 7 PFU per ml, patients 4 through 6 will receive 10 8 PFU per ml, patients 
7 through 9 will receive 10 9 PFU per ml, and patients 10 through 12 will receive 10 10 
PFU per ml. The transbronchial biopsy has been eliminated as part of the evaluation 
because it is not critical in answering questions posed by the protocol. In addition, the 
procedure would probably contribute substantially to the morbidity of the bronchoscopy. 
In response to Dr. D. Miller's concerns regarding helper virus contamination. Dr. Wilson 
noted that all of the vector seed lots have been evaluated by PCR analysis and biological 
amplification assays using the extended culture of the packaging cells. Biological assays, 
using HeLa cells, is the preferable method of analysis. HeLa cells can be diluted to the 
point that no cytopathic effects are observed. These cells can be propagated over a 
period of time. The important issue is what point should the recombinant stock be 
diluted to circumvent the cytopathic effects. Dr. Wilson proposed dividing the lot into 2 
aliquots and evaluate one-half of the lot for replication-competent helper virus. One-half 
of a lot is equivalent to one-half of the maximum dose, i.e., 1 x 10 10 PFU per 10 ml. 
Therefore, the limit of detection would be off by only 1 virus particle. Testing of one- 
half of a lot could be accomplished using approximately 200 10 centimeter plates. 
Dr. D. Miller asked how long these plates will be cultured. Dr. Wilson answered that 
the plates will be grown for 28 days; these plates will be split 2 to 3 times during this 
incubation period. Dr. Post inquired whether the helper virus assay will be performed 
on the seed lot or the production lot of virus. Dr. Wilson stated that the production lot 
will be cultured. The seed lot will be evaluated for other adventitious viruses. A lysate 
will be made from the seed lot and analyzed for gross contamination. 
Dr. Wilson agreed to incorporate all of the suggestions offered by the RAC members 
regarding the informed consent document. He stated that he was especially in 
agreement with the committee members about the issue of fertility. The likelihood of 
germ line transmission is extremely unlikely. The testes of the male primates have been 
extensively analyzed for recombinant protein and RNA expression. Rabbits have 
received large infusions of virus, and their gonads have been evaluated for gene 
expression. No evidence of germ line transfer has been observed in either the primate 
or rabbit models. 
[568] 
Recombinant DNA Research, Volume 16 
