subpopulations will be termed "CD8-enriched cells”. Enrichment will be performed 
prior to transduction of the cellular fractions. 
A minimum of 1.5 x 10^ "CD8-enriched cells" prior to transduction is required. In the 
event of insufficient yield, the donor may be required to return for a second apheresis. 
Non-availability of the donor or a second yield failure may be cause for removal of the 
twin pair from study. 
5.4 Growth and Transduction of T Lymphocytes 
Randomization will take place at this stage, which will determine whether or not the 
CD8-1- enriched cells from a particular donor will be transduced. In each case, half of 
the "CD8-enriched cell" fraction will be frozen and saved as backup (at least 5 x 10^ 
cells). 
The other half of the fractionated cells will be cultured at approximately 5 x 10^ 
cells/well in 24- well tissue culture plates in 100 units/ml of EL-2 (Chiron). Media may 
be supplemented with human AB serum. At the initial plating, 10 ng/ml OKT3 (Ortho) 
monoclonal antibody will be added to each well. The cells will be cultured at 37° C in a 
humidified incubator with 5% CO 2 
Once the T lymphocytes from the donor of the patients randomized to cohorts 1, 2, and 
3 have begun to proliferate (usually 3-4 days after initiation of the culture), vector- 
containing supernatant will be added to the wells after aspirating off the top half of the 
medium. This will be repeated 5 times over 2-3 days. After the final exposure to 
retroviral vector, the genetically altered cells will be purified by immuno-affinity 
techniques (via CD4 receptor). After purification, the cells will be fed with fresh media 
and cultured another 2 to 7 days to permit the cultures to return to exponential growth. 
The percentage of transduced cells will be determined by co-expression of the 
CD8-f-CD4-h phenotype. The required transduction frequency is >1% and with a 
minimum of 1 x 10^ transduced cells after CD4-f selection. If this yield was not 
obtained from the initial transduction attempt or using the backup cells, either the donor 
will be repheresed and the cell processing repeated or the twin pair will be considered 
off-study. 
Once cell volume reaches approximately 400 ml (4 x 10^ cells), the cell cultures from 
all donors will be expanded up to 2 x 10’^ cells employing specialized Life-CellTM 
Tissue Culture flasks which are designed similarly to blood unit bags but are gas- 
permeable and hermetically sealable. It is estimated that the primary expansion will 
require 8-10 weeks. During the expansion steps, it may be necessary to restimulate the 
cells with OKT3 and irradiated syngeneic PBMC. Ten aliquots each containing 
approximately 1.5 x 10^ cells will be cryopreserved in order to provide adequate stock 
cell cultures for subsequent cell expansions for Treatment Period I and II. Prior to 
freezing, an aliquot will be taken for replication-competent retrovirus (RCR) testing. 
Results should be available prior to infusion of cells into the seropositive recipient. 
All cell expansions will be performed with strict adherence to aseptic technique. Cell 
cultures will be tested for sterility employing both aerobic and anaerobic 
microbacteriological procedures. A small sample of expanded T-cells will be analyzed 
by standard flow cytometric phenotypic analysis for CD3, CD4, and CD8 expression. 
Recombinant DNA Research, Volume 19 
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