preparation has been initiated (Carolyn Paradise, M.D., Cetus Corporation). The goal of these 
studies is to use infusion of the exogenous T cell growth factor to induce expansion of the 
patient’s endogenous T cell population and therefore enhanse cellular immune function. 
1 .4 Growth of T Cell Lines from the Blood of Patients with ADA(-)SCID and 
Transduction with the hADA Gene Using Retroviral-Mediated Gene Transfer 
When we began our studies of gene transfer for ADA deficiency we attempted to derive T cell 
lines from the blood of SCID patients in order to have a suitable in vitro system to assess the 
effectiveness of our gene insertion in correcting the primary biochemical consequences of the 
ADA deficiency. Because of the profound T lymphopenia in these patients, we were initially only 
successful in establishing lines when we used the transforming T lymphotropic virus HTLV-I to 
transform the rare T cells found in the blood of patient JF into an immortal cell line. We were 
successful in obtaining three T cell lines (TJF 1, 2, and 3) from this patient. The line TJF-2 
was characterized in detail and had the phenotype of mature T cells (53). In brief, this line 
consisted of a mixture of CD4 and CDS T cells that expressed the IL2 receptor and were 
dependent on \L2 for their growth. Independent clones from the parent line expressed a variety 
of different T cell receptor p chain gene rearrangement patterns indicating that they had a 
unique antigen specificity rather than all being clonally derived from the same differentiated 
precursor cell. Subsequently, we have established and characterized transformed T cell lines 
from 8 additional ADA deficient SCID patients (54) and in each case examined, the lines had the 
phenotype of mature T cells. 
The initial experiments demonstrating correction of the biochemical defect in ADA deficient T- 
cells by the insertion of a functioning human ADA (hADA) gene were performed in these HTLV-1 
immortalized T cells (53,55-57). We demonstrated that the murine retroviral vector, SAX, 
which contains an SV40 promoted hADA gene, could be stably inserted into ADA(-) SCID T cells 
in culture. These SAX-transduced T cells produced enzymatically active hADA at levels 
comparable to normal T lymphocytes in culture. The reconstituted T cells were shown to be 
30-100 fold more resistant to the toxic effects of deoxyadenosine added to the culture medium 
than were the original ADA(-) T cells (Appendix 13.1, Figure 2). Clones were prepared 
from the cultured ADA(-) T cell populations immediately after retroviral vector exposure to 
evaluate them for gene insertion and for receptor heterogeneity. Of the twenty-seven clones 
evaluated by Southern blot, 6 contained an inserted SAX vector (22% successful gene transfer) 
[214] 
Recombinant DNA Research, Volume 14 
