Human Gene Therapy Subcommittee - 11/21-22/91 
selectable marker to HSV-TK. 
Dr. Overell addressed the issue of helper virus contamination. He described the neo 
virus rescue assay that is being used, in which NIH 3T3 cells release replication- 
competent neo R virus when exposed to a sample containing amphotropic helper virus. 
All samples were cultured for 14 days in vitro and then assayed on NIH 3T3 cells; these 
cells were then selected for drug resistance. No neo R colonies resulted, indicating the 
lack of replication-competent virus in these samples. To validate the assay, amphotropic 
helper virus was titered onto NIH 3T3 cells. At a concentration of three focus-forming 
units (FFU) per milliliter, the cells grew to confluency, providing confidence in the 
sensitivity level of the assay. At a concentration of less than one FFU per milliliter, a 
very dramatic growth response was observed with the control. The S + L' assay is also 
used on supernatants from the T cell clones within 2 weeks of infusion. In addition. 
Mink cell focus-forming (MCF) and ecotropic virus assays will be performed. 
In response to Dr. Mclvor's question regarding HyTK retroviral expression, Dr. Overell 
presented Northern blot data in which an SP6 probe specific for the hph segment of the 
HyTK gene was used; the expected transcripts were observed. Dr. Overell described 
Southern blot experiments indicating that the cells contain unrearranged copies of the 
retroviral vector. 
Discussion 
Dr. Kelley asked how insertional mutagenesis would be determined. Dr. Greenberg 
stated that the risk of insertional mutagenesis in the context of serious biologically 
important mutations should be much lower than for gene marking trials, because there 
will be significantly fewer insertion sites (between 5 and 10) as compared to the random 
insertion of marker genes. In turn, the cells can be probed in order to determine 
whether these insertions are biologically important, particularly with regard to 
transformation, because they will be followed for their ability to grow independent of 
growth factors. Therefore, the risk of insertional mutagenesis for this study should be 
extremely low. 
Dr. Erickson inquired about the bystander effect. Dr. Greenberg explained that the effect 
has not been observed and explained in vivo murine data in which the entire lymphoid 
population expresses the TK gene. In this setting, ablating the entire lymphoid 
population with ganciclovir has no effect on other cell populations. Therefore, if 
lymphoid cells are expressing the gene, the bystander effect is probably not going to be 
biologically important. 
Dr. Mclvor asked about the mutation frequency in these T cells clones, citing data from 
the CMV system in which several clones developed resistance to both hygromycin and 
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