the cytotoxic effects of such drugs [57]. This strategy of using HSV-TK as a suicide gene has 
been extensively evaluated in murine models. Administration of ganciclovir to mice bearing 
tumor cells retrovirally infected with the HSV-TK gene or to transgenic mice expressing 
HSV-TK in lymphocytes led to selective elimination of the HSV-TK positive cells with no 
toxicity to normal cells [58,59]. Moreover, the integrated proviral sequences can be detected 
by polymerase chain reaction (PCR) of DNA obtained from PBL to provide a sensitive 
indicator of in vivo persistence following cell transfer. 
The retroviral vector to be used in this study, tgLS +HyTK here referred to as the HyTK 
virus, encodes a suicide gene which is a fusion product of the hygromycin phosphotransferase 
(hph) and HSV-TK genes [60]. This hybrid selectable marker gene encodes a bifunctional 
protein and has the advantage that it confers on transduced cells both resistance to the 
selective drug hygromycin B, and sensitivity to ganciclovir. The HyTK retrovirus has been 
used at a multiplicity of infection of 1:1 to infect human HIV-specific CD8 + T c clones which 
are then selected by culture in hygromycin B. Transduction efficiency of human CD8 + T cell 
clones with the HyTK retrovirus have varied from 0.2 to 2 % as determined by subcloning 
hygromycin B resistant T cells. Such transduced T cells have integrated (he HyTK genome in 
unrearranged form on Southern blot and retain normal function and antigen specificity, but 
are killed in vitro following exposure to low concentrations (0. 1-3.0 jig/ml) of ganciclovir 
[unpublished data]. These concentrations of ganciclovir are not toxic to nontransduced T cell 
clones and are well below those readily achieved in vivo with standard dosing regimens [61]. 
Thus, introduction of the HyTK gene into HIV-specific CD8 + T c clones prior to adoptive 
transfer will provide a marker gene to serve as a sensitive indicator of the duration of in vivo 
persistence of these cells and could provide a novel means of eliminating the clones in vivo if 
toxicity occurs. Preclinical studies in murine models have demonstrated that adoptively 
transferred HyTK transduced murine T cell clones can be ablated in vivo by ganciclovir 
administration (unpublished data). 
The HyTK retrovirus will be produced by Immunex Corporation and has the same structure 
as the LNL6 vector approved for the N2/TIL human gene therapy trial [62], except that it 
contains the HyTK selectable marker in place of the neomycin resistance gene. The 
packaging cell line used to produce the HyTK retrovirus is a clone of infected PA317 cells, 
the same packaging cell line used in the N2/TIL study which has been modified such that it 
does not produce intact replication competent retroviruses (helper viruses) [63]. Moreover, 
both the retroviral supernatant and the transduced HIV-specific CTL clones will be assayed 
for the absence of helper virus using a sensitive neo virus rescue assay prior to using 
transduced cells in immunotherapy. 
Objectives 
A. To evaluate the safety and toxicities of administering increasing doses of autologous CD8 + 
class I MHC-restricted HIV-specific T. clones transduced bv retrovirus mediated gene transfer 
to express a marker/suicide gene. 
B. To determine the duration of in vivo survival of adoptively transferred HIV-specific T. clones. 
C. To determine if ganciclovir or acyclovir therapy can efficiently eradicate the genetically 
modified adoptively transferred T cells. 
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Recombinant DNA Research, Volume 15 
