proliferation. If toxicity or suspicion of inappropriate gene expression occurs, the drug GCV can be 
stopped thus immediately limiting any negative side effects. 
6. Psychological and social implications. 
It is likely that the initiation of these experiments will be associated with tremendous 
attention by the media. We will follow a strict code of confidentiality as mandated by the R.A.C. and 
inform the patients of this potential problem. 
7. Risks to Others 
The only potential risk to others is the possibility that the recombinant virus could be transmitted 
to another individual, take up residence, and cause disease. We believe this is highly unlikely for a 
variety of reasons. 
One very important aspect of this scenario relates to the likelihood that transmission of Ad.RSVf/c 
will cause disease. We obviously do not think this is likely based on the fact that we are proposing to 
use it therapeutically. Toxicity could be due to one of two mechanisms: expression of HSVf/c or 
pathology due to the inherent properties of Ad5. As we argued above, ectopic or unregulated 
overexpression of HSVf/c without administration of GCV does not appear to be toxic. Furthermore, 
pathology of the recombinant due to its properties as an infectious virus is unlikely because all 
patients will have had previous immunity to the virus and the recombinant should have a tremendous 
growth disadvantage due to the deletion of El . Another possibility is that the Ad.RSVf/c will undergo a 
recombination with homologous wild type Ad to form a more toxic structure. There is no reason to 
expect this to be more pathogenic than either parent. 
The next consideration relates to the probability that the Ad.RSVf/c virus can replicate, spread and be 
transmitted. We believe that this will be unlikely. In order to assure that this does not occur we 
will keep the patients in isolation for a period of 10 days following the treatment. The patient's 
nasal swabs and sputum will be evaluated for Ad.RSVf/c sequences before discharge. If there is 
evidence of ongoing spread the patient will be asked to stay in the hospital for additional days. We 
think this will be extremely unlikely. The other possible situation that could lead to spread is if the 
patient becomes superinfected with a homologous wild type virus which mobilizes the recombinant 
genome. It is impossible for this to occur through the generation of a replication competent virus 
containing the minigene due to size limitations of packaging. The spread of the genome would have to 
occur by adenoviral functions provided in trans. We think this is highly unlikely and should not 
prevent the study. In order to address this potential concern we will work with the referring 
physician to be particularly cognizant of signs and symptoms characteristic of adenoviral infections 
and will continue to survey nasal swabs for Ad.RSVf/c sequences using the PCR assay. 
V.D. AdRS WTK Virus Formulation (For Details see Appendix G) 
Similar to the generation of recombinant retroviral gene therapy reagents, adenoviral reagents have 
been constructed to provide "cassette" oriented approaches to their production. A recombinant 
adenoviral vector was constructed from a modified adenovirus type 5 in which the minigene of 
interest is inserted in place of El a and Elb. This vector is cotransfected along with the large 
fragment of the enzyme-restricted Ad5 DNA into 293 cells. 293 cells are a human kidney cell line 
that contains a functional El a gene and provides a trans-acting El a protein in order to allow for 
homologous infectious virions and formation of plaques. A Master Cell Bank (MCB) of 293 cells 
have been previously established and evaluated for performance, in terms of production of 
recombinant adenoviruses and for the absence of other pathogenic contaminants. Individual plaques 
are isolated and functional DNA would be identified by restriction cleavage and southern blot 
analysis. The MCB is then infected with the crude viral lysates and plaque-purified a second time in 
order to generate a seed lot using 293 cells. The purified seed lot lysate is subjected to safety 
testing. The MCB is then plated again and infected with the certified viral seed lot. Lysates are 
harvested from the infected cells and virus is purified from the lysate and cryopreserved. 
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