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 analyzed for 
the above features. 
An aliquot of the cells infused into the patient will be saved and 
Southern analysis will be performed 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. Probing for T 
cell receptor beta chain gene rearrangements will also be done to 
address clonality with respect to T cell specificity (38). Cells will also 
be tested for replication competent retrovirus by S+L- with 3T3 
amplification. 
Two retroviral vectors, each containing the neoR gene, will be used as 
markers. These retroviral vectors are designated LNL6 and GINa. 
They consist of the following elements listed 5' to 3': Moloney murine 
sarcoma virus long terminal repeat (LTR) promoter, psi sequence, 
polycloning site, neomycin phosphotransferase gene, polycloning site, 
and Moloney murine leukemia virus LTR. Both are produced as FDA 
certified material by Genetic Therapy, Inc. (Gaithersburg, MD) which 
has manufactured the vectors used in the human ADA deficiency gene 
therapy, human melanoma TIL, and human bone marrow gene 
marking protocols. Both vectors have been previously used in other 
human gene transfer experiments. They consist of high titer (105 -106 
colony forming units per ml) supernatants of packaging cell lines. The 
supernatants are free of pathogens and helper virus. The retroviral 
vectors are capable of a single infection; i.e., they are replication 
incompetent. The neomycin phosphotransferase protein they encode 
remains an intracellular protein. 
One vector will be used to mark CD4-enriched cells in a patient, while 
the other vector will be used to mark CD8-enriched cells. Each vector 
contains unique sequences which will allow one to discriminate 
between them using PCR based Southern analysis using radiolabeled 
probes corresponding to the neomycin phosphotransferase gene 
which is found only in the transferred gene sequences but not in the 
native human genome. LNL6 and GINa differ in large part only by the 
length of noncoding sequences 3' to the neoR coding sequence. Use 
of PCR primers which flank this insert will generate fragments which 
differ in size. Using the primers 5' I I 1 TGTCAAGACCGACCTGTCC3’ 
(homologous to positions 1643-1664 in LNL6, and to 1658-1679 in 
GINa) and 5TTTCATTCCCCCC l I I I ICTGG3’ (antisense to positions 
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