Recombinant DNA Advisory Committee - 09/14-15/92 
addition of the culture supernatant. Culture supernatant from LNSX transduced cells 
had no effect on the growth on nontransduced cells. Additional evidence of the 
bystander effect is seen in the morphologic changes observed in nontransduced tumor 
cells. Untransduced tumor cells grow in a three-dimensional configuration, not in 
monolayers as is observed with normal cells. However, when untransduced tumor cells 
are cultured with the p53 retroviral vector supernatant, morphological events occur 
similar to those of programmed cell death and apoptosis, i.e., formation of blebs or 
vacuoles. This observation is in contrast to the appearance of cells that normally express 
p53. The extreme morphological changes observed when cells are transduced with p53 
may correlate with the release of factors into the supernatant. 
Dr. Roth responded to Dr. D. Miller's question regarding the stability of the viruses and 
potential for oncogenic transformation. Both the H358a and H460a cell lines have been 
maintained in culture for as long as six months that have been transduced with both the 
p53 wild-type and the antisense K-ras genes, and these genes have been continuously 
expressed with no evidence of oncogenic transformation. Dr. D. Miller stated that if 
these cells contained a mutant p53 gene, the mutation would not be detected. The usual 
assay for transforming virus within a preparation is to perform an assay on a monolayer 
cell line. Dr. Roth said that these experiments had been performed and that no focus 
formation has been observed when one milliliter of supernatant containing 10 7 CFUs 
were cultured with NIH 3T3 cells for three weeks. The same results were observed for 
both vectors. Dr. D. Miller asked if the wild-type K-ras virus was used as a positive 
control for these experiments. Dr. Roth said that this control was not included. 
Dr. Roth responded to questions regarding transduction efficiencies. The packaging cell 
line currently being used produces viral titers of greater that 10 6 CFUs, without the 
addition of co-cultivation techniques. Semi-quantitative PCR demonstrates between 30 
and 50% transduction. The protocol specifically states that patients will not be treated 
unless this level of virus production is obtained. GTI will be producing the retroviral 
supernatant and performing the necessary quality control measures. MD Anderson's 
IRB has already approved the LNSX vector for use in the clinical trial; therefore, LNSX 
will be the initial vector. In the event that a superior vector is identified, a request will 
be made to the RAC for a minor modification to the protocol. 
Regarding possible toxic side effects of the protocol, Dr. Roth stated that no toxic effects 
have been observed in mice that have been injected with retroviral supernatant 
containing 350 times the number of viral particles proposed for the human protocol. 
Some of these animals have survived for six months with no specific manifestations of 
acute toxicity. 
Dr. Roth stated that the MD Anderson IRB has consulted with Dr. Goodrich, an 
infectious disease expert. Both Dr. Goodrich and the IRB have concluded that the 
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