analyzed for the above features and cryopreserved for future patient infusion. 
In part 2.A., following transduction the cells will be allowed to regain exponential growth and ' 
then will be placed under selection with 0.15-1.0 mg/ml G418 and/or 200-1200 |iM 
2’deoxyadenosine for 1 to 14 days. The optimal conditions for selection will have to be 
established for each patient. The cells will be selected until the ADA level in the cultured cells 
reaches at least 80% of the level of ADA in normal blood MNC. At this point, approximately 
80% will be infused into the patient and the remainder will be returned to culture and expanded 
in number for possible future use in part 2.B. The cells will then be subcultured every 2-8 
days as needed and transferred into 6 well plates, flasks, gas permeable bags and/or a hollow 
fiber culture system as the cells expand in number by techniques similar to those used in the 
previously approved N2/TIL protocol 86-C-183. Again, conditions may be modified by the PI 
to take advantage of technical advances or to optimize results for an individual patient. 
In part 2.B. of the protocol, the ADA gene-transduced, selected T cell population will be selected 
to enrich for cells expressing the introduced genes as described above and will be expanded in 
number so that as many as 3x10^/kg are available for infusion. Mixtures prepared from 
cryopreserved aliquots of earlier cell cultures may be made to broaden the infused repertoire 
should the cultures appear to have become oligoclonal based on the pattern of TCRp chain gene 
rearrangements in the cultured populations. 
An aliquot of the cells infused into the patient will be saved and we will subsequently perform 
Southern analysis on the DNA from the cultured cells after digestion with a restriction 
endonuclease which does not cut within the vector sequence to determine whether the gene- 
modified cells are polyclonal with respect to retroviral insertion. We will also probe for T cell 
receptor p chain gene rearrangements to address clonality with respect to T cell specificity as 
we have done in the N2/T1L protocol. (69) Cells will also be tested for their responses to IL-2 
withdrawal and for replication competent retrovirus by S+L- with 3T3 amplification. 
6.3 Reinfusion of hADA Transduced T-Lymphocytes 
The transduced cells will be harvested, washed and resuspended in normal saline. The final cell 
preparation will be filtered through a platelet filter and transferred into a syringe or 
transfusion pack for infusion. A test dose of 2-5% of the total volume will be infused by 
Recombinant DNA Research, Volume 14 
[227] 
