Human Gene Therapy Subcommittee - 11/21-22/91 
gene can be positively selected by hygromycin B or negatively selected by a drug such as 
ganciclovir. 
Dr. Mclvor stated that there are significant in vitro data to support the thesis that the 
herpesvirus TK gene is an ablatable function. However, he inquired as to the 
comparative effectiveness of the fusion protein as compared with the natural TK gene. 
What biochemical studies have been performed to study the function of the fusion 
protein? Does expression of the HyTK fusion protein confer ganciclovir sensitivity to the 
same extent as the natural TK gene? Has HyTK been tested in vivo? The natural 
mutation frequency with which these cells will lose TK function could be greater than 1 
in 10 6 cells. The assay methods proposed by the investigators are not sensitive enough to 
quantitate mutations at this level. He suggested that a clonal system should be used to 
determine the level of ganciclovir insensitivity in the HyTK transduced cell population. 
Dr. Mclvor stated that the HyTK retrovirus was similar to the LNL6 retrovirus 
previously approved by the HGTS and the RAC. Since several envelope sequences are 
missing from the HyTK vector that are present in LNL6, it may be a safer vector. 
However, since this retroviral construct is new, the investigator is obligated to provide 
data demonstrating the absence of replication-competent virus as well as other infectious 
agents. 
Dr. Mclvor discussed the transduced cell population. Lymphocytes will be obtained from 
the patient and cloned in vitro. The cloned lymphocytes will be subsequently transduced 
with the HyTK vector and subcloned. He cautioned the subcommittee that this highly 
selected population may behave differently from the parent lymphocyte population, and 
that the subcommittee should consider whether any additional risk will be imposed on 
the patient. 
Dr. Mclvor inquired about the effect of azidothymidine (AZT) administration on 
hematopoietic engraftment post-transplant. The investigators cited data demonstrating 
that HIV-specific T cell clones were generated; however, other than one Southern blot, 
there are no data to substantiate the preclinical results. Have these T cell clones been 
transduced with the HyTK virus and expanded? What is the frequency of gene transfer 
in these clones? He asked Dr. Greenberg to address transduction expression and 
ganciclovir sensitivity in the T cell clone population. What is the effect of HyTK 
transduction on the immunological function of the T cell clones? Dr. Mclvor asked Dr. 
Greenberg to explain the choice of primers that they have chosen to use for PCR 
amplification. Can assays be performed to distinguish among different T cell clones? 
Regarding ganciclovir administration, Dr. Mclvor noted that ganciclovir may be given for 
purposes other than T cell clone ablation, such as for cytomegalovirus infections or 
prophylaxis. He asked the investigators to explain when ganciclovir would be 
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