Finally, we will assay for gag expression in the expanded cells by a commonly used and 
highly sensitive HIV-1 p24 antigen capture assay (Coulter). Other novel reverse- 
transcriptase inhibitors and antivirals (27, 54) have potential application to this 
problem in future protocols. 
1. Harvesting of patient PBMC for ex vivo stimulation 
Following completion of baseline data acquisition, study subjects will undergo 
leukopheresis or phlebotomy of not more than 8 ml/kg per 4 week interval. Fresh 
peripheral blood mononuclear cells will be separated from erythrocytes and neutrophils 
by Ficoll-Hypaque density gradient centrifugation. The PBMCs will then be washed, and 
depleted of CD8+ cells utilizing murine anti-human CD8-monoclonal antibody coated 
flasks (Applied Immune Sciences CELLectorTM Flasks). Each flask will be loaded with 30 
mis of 2-3 x 10 cells/ml and incubated at room temperature for one hour. Non- 
adherent cells are to be stimulated in CM with 10 ng/ml OKT3 antibody [CM = AIM-V 
serum-free media (Gibco) with 2mM glutamine, 50 U/ml penicillin, 50 ug/ml 
streptomycin, 2.5 ug/ml Fungizone, IL-2 1000 U/ml and the HIV-1 reverse 
transcriptase inhibitor nevirapine (Boerhinger-Mannheim) at 40 nM and CD4- 
pseudomonas exotoxin (Upjohn)]. Cellular phenotype is assessed by flow cytometry 
prior to expansion. 
2. Expansion in IL-2 . OKT3-activated cells are resuspended in fresh CM containing 60 
U/ml of IL-2 (Cetus, Emeryville, CA) at 1-2 x 10 5 cells/ml and expanded in 3000 ml 
culture bags (Lifecell PL732, Fenwal, Deerfield, IL) each containing about 500-1000 
ml media. Cells are grown to maximum density (about 1-2 x 10 6 cells/ml). Expansion 
is estimated to take 3-4 days. 
3. Transduction . Two identical aliquots of cells will be transduced simultaneously, one 
with the ribozyme-bearing vector and with the control vector (vector lacking the 
ribozyme). Frozen viral supernatants will be stored at -80°C. On the day of 
transduction, aliquots will be thawed and passed through 0.45 micron filters. Cells will 
be resuspended at a concentration of 10 5 per ml in a transduction mixture having 50% 
CM and 50% viral supernatant (MOI of 1 to 3) supplemented with protamine sulfate 
(49) at a final concentration of 5 pg/ml. After 4-8 hours incubation at 37 degrees C, 
cells will be washed 3 times in CM and introduced into tissue culture bags for large scale 
expansion for therapy. 
[392] 
Recombinant DNA Research, Volume 18 
