Recombinant DNA Advisory Committee - 12/1-2/94 
Dr. Chiang explained that this modified vector incorporates a 3’ non-coding HSV-TK deletion which 
circumvents any potential for RNA splicing and results in higher vector titers. In response to Dr. Parkman’s 
question regarding the necessity for VPC, Dr. Chiang stated that both VPC and GCV are essential for the full 
therapeutic effect. Dr. Chiang described an experiment that was conducted to determine the relationship 
between HSV-TK enzyme activity and the "bystander" effect. The 9L glioblastoma cell line was added to: (1) 
VPC expressing variable levels of TK enzyme activity, (2) PA317 (some endogenous TK activity), and (3) 
NIH3T3TK(-) (no endogenous activity). Data demonstrate that a threshold level of TK activity is required for 
the GCV "bystander" effect and that little difference is observed beyond this threshold level Dr. Parkman 
commented that the experiment described by Dr. Chiang involved a glioblastoma cell line, not a human 
squamous cell carcinoma line. 
Dr. Chiang presented data demonstrating PA317 (endogenous TK) sensitivity to GCV killing. In contrast, 
NIH3T3TK' and untransduced 9L cells were insensitive to GCV killing. She noted that similar in vitro studies 
have been conducted using a squamous cell carcinoma cell line; however, the in vivo experiments involved only 
the hepatocellular carcinoma model 
In response to Dr. Brinckerhoffs concerns regarding immune reactions. Dr. Chiang stated that VPC immune 
responses were observed in the rat model; however, no serious adverse effects were identified upon 
pathological examination. 
In regard to Dr. Miller’s question about VPC persistence, Dr. Chiang said that no primate data is available. 
Dr. Anderson stated that he has conducted experiments in which VPC were injected into porcine livers and no 
complement lysis was observed for 5 days. Porcine complement lyses murine VPC in vitro, which indicates 
that the complement levels within the liver are low. VPC are lysed within 30 minutes when injected directly 
into the blood; however, VPC will remain viable up to 5 days in the lymphatic system. 
Dr. Stambrook presented data demonstrating the "bystander" effect in a squamous cell carcinoma cell nude 
mouse model The first experiment involved mixing tk+ and tk- squamous carcinoma cells at a 1:3 ratio and 
injecting them into the left flank of the mouse; an equal number of tk- cells (5 x 10 6 ) were injected into the 
right fl ank. Following a period to allow for tumor growth, treatment with GCV was carried out, and both the 
right and left flanks showed tumor regression. Upon histological examination of the regressing tumor, there 
was some evidence of lymphocyte infiltration. The significance of this observation remains to be determined. 
In the second experiment, squamous cell carcinoma cells were injected in both right and left flanks of the 
mouse, followed by injection of PA317 cells; following treatment with GCV, there was tumor regression in 
both flanks. A third experiment was conducted as a control; when HSV tk- 3T3 cells were used instead of 
PA317 cells, there was no evidence of tumor regression following GCV treatment. This result was consistent 
with the suggestion of Dr. Parkman that PA317 cells may contain HSV-tk. 
Dr. Parkman noted that the investigators may have discovered a new mechanism by which TK and GCV may 
be able to destroy distant tumors, but it seemed unlikely that the killing effect was the same as the "bystander" 
effect observed by other investigators. For example, it was unlikely that there would be any gap junction 
established between human and murine cells. Furthermore, there was no data to suggest the persistence of 
the VPC for a sufficient period of time in vivo to allow for gene transduction to occur. Additional experiments 
to explain the "bystander" effect would be necessary. 
Dr. Brinckerhoff agreed with Dr. Parkman. She was concerned about the lack of immunological data in 
humans. The data from the rat model does not adequately address the question of immune reaction. If an 
anti- VPC reaction does occur, these cells could be lysed even more quickly by complement. 
Committee Motion 
A motion was made by Dr. Parkman and seconded by Dr. Erickson to defer the protocol. Although the 
proposal is a logical extension of the HSV-TK/GCV strategy previously used in the brain tumor protocols, too 
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Recombinant DNA Research, Volume 20 
