b. Direct toxicity of the virus to the lung or chest wall : It is theoretically possible that the 
adenovirus could induce direct toxicity to the cells of the lung or pleura. We saw no direct evidence 
of this in our mouse or rat, thus think this unlikely. 
c. Replication of the virus and expression of viral proteins : The virus has been engineered in a way 
that the region responsible for activating its transcriptional units (El A), has been deleted. In 
principal, therefore, the recombinant genome should not replicate or express viral proteins in the 
recipient cell. In practice, certain cell types under special conditions can transactivate the viral 
genes using endogenous cellular factors. 
We have established a model of malignant mesothelioma growing in SCID mice as a way to assess the 
therapy in a relevant biological setting. This model was used to evaluate the possibility of viral 
protein expression and replication of the El deleted HSVf/c virus in the context of a human tumor. 
These studies failed to detect viral protein expression or ongoing replication of the virus in human 
xenografts that were stably expressing the recombinant gene, although it must be acknowledged that 
the adenovirus does not replicate well in the mouse. The recent studies from Crystal's group are 
relevant to this point. In these studies Setoguchi et al. (1994) used adenovirus to transfer the ocl- 
antitrypsin gene to the peritoneal cavity of cotton rats, a species highly permissive for the 
replication of adenovirus. They detected no expression of the transgene (as assessed by PCR) in any 
organ within or outside the abdomen. 
The recent studies of the Wilson group using adenovirus transfer of the CFTR (cystic fibrosis 
transmembrane conductance regulator) gene in non-human primates and early human trials 
confirm the relative safety of this approach to thoracically directed gene transfer (Simon et al., 
1993). Replication competent viruses have not been detected. 
It is important to consider the consequences of the unexpected dissemination of the recombinant 
virus or a replication competent derivative. Previous experience with adenoviruses in humans 
suggests that they are relatively benign. Adenoviruses are endemic in the population causing a 
variety of self limited infections. Infection with Ad5 causes mild respiratory infections 
characterized by coryza, pharyngitis, fever, malaise, and tonsillitis. The relative safety of 
adenoviruses was evaluated in a series of experiments, performed in the late 1960's and early 
1970's, in which volunteers were exposed via the respiratory tract to wild types forms of 
adenoviruses including the serotype used in this study (Ad5). 
d. Oncogenesis: An additional consideration relates to the malignant potential of these gene transfer 
reagents. In contrast to retroviruses which integrate into the genome, adenoviruses in general are 
maintained in the genome as extrachromosomal elements which theoretically cannot cause a 
secondary malignancy due to insertional mutagenesis. In support of this, is the fact that no human 
malignancy has been directly linked to adenoviruses of this serotype. 
e. Virus Mediated Cellular Immune Response : An important characteristic of currently available 
recombinant adenoviruses is that gene expression is relatively transient and inflammation occurs at 
the site of gene transfer. Although these represent clear limitations with regard to treatment of gene 
deficiency syndromes, it is currently unclear how these characteristics will impact on either 
toxicity or efficacy of cancer treatment. We will carefully monitor the patients for evidence of an 
inflammatory response in the pleural space. If we detect an increase in pleural fluid, signs of 
pleuritis or fevers, a thoracentesis will be performed with subsequent analysis of the fluid for 
evidence of acute (polymorphonuclear) or cell mediated (lymphocytic) inflammatory response. 
5. Toxicity of the HSVtk gene 
The HSVf/c gene has no direct cellular toxicity by itself, without co-administration of the drug 
GCV. The mechanism of action is related to competitive inhibition of purines during active cell 
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Recombinant DNA Research, Volume 20 
