Recombinant DNA Advisory Committee - 3/3-4/94 
susceptible to viral integration in the 24 hour period of treatment. What percentage of 
cells in human tumors will be in cycling? Dr. Roth answered that cell cycling problems 
will be overcome by administering repeated injections. Since the bulk of the tumor will 
be removed by laser treatment, a multiplicity of infection of 5 viral particles to one 
tumor cell is achievable. 
Responding to the question of the size of DNA fragments in the Southern blot analysis 
of cells transduced with the LN/?5JB vector, Dr. Roth said that digestion with the Kpn 
restriction enzyme should yield a fragment approximately 8.7 kb. Ms. Grossman 
questioned the validity of the Southern blot data submitted by Dr. Roth. This data 
shows that DNA fragments of the same size of 10.8 kb are detected in cells either 
transduced by the LNSX vector or by the LNp5JB construct with gene insert. Dr. Roth 
said that he was not sure why the DNA fragment migrates aberrantly at this point. Dr. 
Straus said that the DNA fragment with the p53 gene insert should not migrate at the 
same rate as the fragment obtained from the vector itself. Dr. Roth said that the present 
1% gel analysis will not permit resolution of this difference. Drs. Straus, Post, and Ms. 
Grossman said that the size difference should be approximately 5.5 kb, and it should be 
resolved in this gel analysis. Ms. Grossman asked whether Northern blot analysis of 
vector RNA in transduced cells has been performed in order to determine whether there 
is any vector structure rearrangement. Dr. Roth referred to a letter by Dr. Harry 
Findlay of Emory University, Atlanta, Georgia, which states that the transduced p53 
expression was detectable in his Northern blot analysis. Ms. Grossman said that the data 
is not presented to answer her question on vector rearrangement. Ms. Grossman 
remarked that from her understanding of the situation that every time the principal 
investigators at Genetic Therapy, Inc., Gaithersburg, Maryland, have made a production 
run on this virus, they found vector rearrangement. Dr. Roth said that they are using a 
different vector construct and a different producer cell line. Ms. Grossman said that 
vector rearrangement is an important issue to be resolved before proceeding to human 
trials. She said that the data presented is not convincing. 
Responding to questions about the "bystander" effect. Dr. Roth presented data reported 
in his published paper entitled: A Retroviral Wild-type p53 Expression Vector Penetrates 
Human Lung Cancer Spheroids and Inhibits Growth by Inducing Apoptosis, published in 
Cancer Research (Vol. 53, pp. 4129-4133, 1993). This data demonstrated that substances 
released from p55-induced apoptosis inhibit tumor cell growth. Dr. Roth said that very 
recent data suggested that this substance may be a protein molecule since its activity is 
destroyed by protease digestion. This observation may offer a possible explanation for 
the mechanism of the "bystander" effect observed in the animal model. 
Dr. Roth said that he is unable to perform the cell mixing experiment to demonstrate 
that cells transduced with p53 are capable of inhibiting the growth of untransduced cells. 
Cells transduced with the p53 gene construct undergo apoptosis, and they cannot be 
established as a cell line. 
Responding to Dr. Parkman's question on in vivo transduction efficiency. Dr. Roth 
presented data demonstrating approximately 60% transduction efficiency in an orthotopic 
Recombinant DNA Research, Volume 19 
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