5.0 Stratification and Randomization 
Not applicable 
6.0 Nature of Procedures or Therapeutic agents 
6.1 Isolation and Culture of Peripheral Blood Mononuclear Cells from the Patient 
The child will be admitted to the Clinical Center of the National Institutes of Health. A 
phlebotomy or lymphopheresis (performed by the NIH Apheresis Unit according to age- 
appropriate procedures following the previously approved Metabolism Branch Protocol 82-C- 
44) will be used to obtain lymphocytes. 7 ml of blood/Kg will be sampled per leukaphoresis 
and a maximum of 7 ml/kg will be obtained by phlebotomy every 4 weeks. Fresh peripheral 
blood mononuclear cells (MNC's) will be separated from the red cells and neutrophils by 
Ficoll-Hypaque density gradient centrifugation. The MNC’s will then be washed, counted and 
cultured at approximately 1x10® cells/well in 24 well tissue culture plates in AIM-V* which 
consists of AIM-V (GIBCO) with 2 mM glutamine, 50 U/ml penicillin, 50 |ig/ml streptomycin, 
2.5 pg/ml Fungizone and 25-1000 units/ml of IL-2 (Cetus). 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% CO2. The conditions of culture and lymphocyte stimulation 
may be modified by the PI during the course of this protocol to take advantage of improvements 
in technique or media. 
6.2 Growth, Transduction and Selection of ADA-Deficient T-Lymphocytes 
Once the T-lymphocytes have begun to proliferate (usually 24-96 hours after initiation of the 
culture), 1_ASN vector-containing supernatant (containing protamine 5-10 pg/ml and up to 
1000 U/ml IL2) will be added to the wells after aspirating off the top half of the medium. This 
will be repeated 1-2 times daily for a period of up to 7 days. After the final exposure to 
retroviral vector, the cells will be fed with fresh AIM-V* and cultured another 2 to 7 days to 
permit the cultures to return to exponential growth. In part 1, approximately 80% of the 
culture will then be infused into the patient and the remaining cells returned to culture for 
continued growth and selection procedures, and/or analyzed for phenotype, T cell repertoire, 
and percentage of cells demonstrating vector integration. Selected cultures will be periodically 
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