harvest will be processed to remove contaminating red 
cells. Enrichment for CD34+ cells will be performed 
using the CellPro Stem Cell Separator. This procedure 
involves the incubation of the cells with a 
biotinylated murine anti-human CD34 antibody 12.8, 
washing the cells, and passing them over an avidin 
Biogel column. The cells adhering to the column will 
be collected after mechanical dislodgement . After 
enrichment, a sample of CD34+ cells will be saved for 
further analysis and use as pre-gene therapy controls 
for colony- forming and other assays. 
3.33 Transduction and Transplantation Procedures : 
The retroviral vector supernatant for this study will 
be produced by Genetic Therapy, Inc. (GTI) . GTI has 
produced the retroviral vector supernatants used in 
most of the gene therapy studies performed thus far. 
The vector supernatant used in this protocol will be 
produced and quality control- tested in a similar 
fashion as for other retroviral vector protocols. 
1 . Transduction Procedure 
After removal of aliquots of enriched cells for 
studies, the remainder of the CD3 4 -enriched 
population of cells will be transduced with the 
retroviral vector in flasks. Separated CD34- 
enriched cells will be cultured (1 x lOVnil) in 
vector supernatant with IL-3, IL-6, and SCF and 4 
/ig/ml protamine sulfate for a period of 72 hours. 
Every 24 hours the cells will be pelleted and 
resuspended in fresh supernatant, growth factors, 
and protamine. After the period in culture, the 
cells will be washed and samples taken for 
analysis (gene transfer efficiency, colony- forming 
ability, assays for replication- competent 
retrovirus) . The remaining cells will be 
resuspended in heparinized medium for reinfusion. 
Included in Appendix VI is a sample certificate of 
the tests that will be performed on each 
hematopoietic stem cell sample prepared for 
reinfusion into the patient. 
2 . Reinfusion of Transduced Cells 
The transduced cells will be reinfused into the 
patient over a period of two hours or less. 
Premedication, given 30 min before infusion 
begins, will consist of 25 mg diphenhydramine IV 
and 650 mg acetominophen given po (100 mg 
hydrocortisone IV is optional) . Vital signs will 
be monitored every 15 min up to two hours post- 
infusion. 
3 . Post- Infusion Care 
Initially, patients will be monitored in the 
hospital overnight. If no adverse events are 
seen, the patients will be discharged the next day 
for subsequent outpatient follow-up. 
4 . Frequency of Infusion of Transduced Cells 
Patients may recieve up to four cycles of 
transduced PBSC. The cycles will be no more 
D 
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