and each of these SAX vector-containing clones produced hADA (Appendix 13.1, Figure 3). 
Thus the principal biochemical defect in ADA(-) SCID T-cells was efficiently corrected with 
retroviral-mediated gene transfer. 
The ability to more readily recover T-lymphocytes from ADA deficient children on PEG-ADA 
treatment has allowed us to evaluate gene transfer and cell function in vitro without the 
necessity of prior cell transformation with HTLV-I. After testing a variety of culture 
conditions, stimulants and growth factors, we found that we were able to successfully grow 
large numbers of T cells in vitro from these patients. The optimal culture conditions defined to 
date includes AIM-5 medium supplemented with 50-1000 U/ml rlL2 and stimulated with the 
anti-T cell receptor monoclonal antibody OKT3. We have established non-transformed T cell 
lines from 5 patients and in 4 of these we have been able to expand these cells over 1000 fold in 
culture in a 3-5 week period. 
We have tested a variety of vectors that carry the hADA cDNA driven by different promoters for 
their ability to express hADA in immortalized human lymphoid and myeloid cells (58). The 
promoters used include a human cytomegalovirus immediate early promoter (vector LNCA), the 
simian virus 40 (SV40) early promoter (vector LNSA), a promoter containing the 
lymphotropic papova virus enhancer (vector LNLA), the beta-globin promoter (vector 
LNBBA), and the Moloney murine leukemia virus promoter located in the retroviral LTR 
(vector l_ASN). The vector LNSA is similar in design to the SAX vector described above. Of all 
these vectors, the vector LASN directed the highest level of hADA expression when transferred 
into the DHL-9 human lymphoblastoid line (4 p.M/hr/mg protein), a level at least comparable 
to ADA levels in normal lymphoid cells (0.3-0.5 pM/hr/mg)(9,12,59). The vector LASN 
contains the hADA [A] gene promoted by the 5’ LTR [L] and a neomycin resistance [N] gene 
(Neo*^) promoted by an internal SV40 promoter [S]. When inserted into murine bone marrow 
cells, expression of hADA could be detected in recipient mice for 6 months after cell transfer 
(60). See Appendix LASN. 
The hADA gene was inserted into exponentially growing non-transformed ADA(-) T cells by 
using the vectors that were the most active in human hematopoietic cell lines; SAX, LASN, 
LNSA, and LNCA. The latter three vectors were derived from the same vector construct as the 
LNL6 vector used in our N2/TIL human protocol. These vectors are packaged in the 
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