OCT-28-94 09 . 06 FROM ■ USC DIV. HEMOTOLOGY 
ID-213 224 6987 
PAGE 
7. Gene expression was determined by growing CFU-GM colonies in methyl- 
cellulose in the presence and absence of G418. The number of colonies that 
expressed the gene was determined by the number of colonies grown with G41 8 
divided by the number of colonies grown without G41 8. 
8. Contained in the Drug Master File supplied to the FDA by GTI. 
9. Contained in the Drug Master File supplied to the FDA by GTI. 
1 0. Contained in the Drug Master File supplied to the FDA by GTI. 
C. CLINICAL PROTOCOL 
1 . The in vitro system that determined the preclinical efficacy was as follows: bone 
marrow or PBPC samples were enriched for CD34+ cells by positive 
immunoselection with anti-CD34 antibodies using an immunoabsorption column 
(CellPro Inc.). After incubation with biotinylated anti-CD34+ antibody, the cells 
were passed over an avidin-conjugated column and the CD34+ cells bound to the 
column were removed by mechanical agitation and collected. The cells were 
exposed to the LNL6 or G1 Na retroviral vector-containing supernatants prepared 
by Genetic Therapy Inc. The CD34+ enriched cells were preincubated for 42 
hours in the presence of long IL-3, 50ng IL-6 and 10% autologous plasma at 37° 
C. After washing, the cells were incubated at 37°C for six hours with lOng IL-3, 
50ng IL-6. protamine sulphate and the appropriate vector-containing supernatant 
at a ratio of approximately 10:1 (vector partides: cells). The transduced cells were 
then grown in methyl cellulose In a standard CFU-GM assay in the presence and 
absence of G418. Genomic DNA was obtained from individually picked CFU-GM 
colonies and the marker gene was determined by PCR with oligonucleotide 
primers homologous to the Neo* gene vectors: GINa: sense 
5'GGT GGAGAGG CTATTCGGCTATGA 3\ antisense 5” AT CCT GAT CGACAAG ACCGGCTTC 3‘. 
LNL6; sense sr caagatggattgcacgcagg 3*. antisense oocgctcagaagaactcgtc 
3’. Each thermocyde consisted of denaturation (94°C, 1 min), annealing (51°C, 3 
min), and polymerization (72°C, 3 min) for 30 cydes followed by 10 min at 72°C. 
The PCR products were electrophoresed on agarose gel, stained with ethidium 
bromide and visualized by UV light. 
2. The end points of the protocol are: 
a. Detection of LNL6 and/or G1 Na gene marker in hematopoietic precursors and 
mature cells in patients who undergo autologous transplantation of CD34+ 
enriched bone marrow cells marked with one vector and CD34+ enriched PBPC 
marked with the other vector. 
b. Detection of LNL6 and/or GINa gene marker at sites of relapsed breast cancer 
or lymphoma In patients who undergo autologous transplantation of CD34+ 
enriched bone marrow cells marked with one vector and CD34+ enriched PBPC 
marked with the other vector. 
(534] 
Recombinant DNA Research, Volume 20 
7/20 
I 
