packaging cell line (25), generates infectious SAX virus at concentrations as 
high as 6.0 x 106 neo^ cfu/ral. Cocultivation of cynomolgus monkey bone narrow 
mononuclear cells with an S3A monolayer for periods ranging from 4-24 hours 
conferred resistance to 1.7 mg /ml G418 in a maximum of 7% (at 24 hrs.) of the 
CFU-C detected on day 14 (Fig. 2, curve B). In the second protocol, bone 
marrow cells were exposed to virus-containing supernatants derived from the S3A 
producer cells, using ratios (SAX virustbone marrow mononuclear cells) of 
between 5 and 10 to 1. After 2 hours of exposure, followed by extensive 
washing with phosphate-buffered saline (FES), over 10% of day 7 CFU-C (data not 
shewn) and 28% of the day 14 CFU-C (Figure 2, curve C) detected were resistant 
to toxic concentrations of G418. 
In Vivo Studies 
The in vitro results outlined above were subsequently supported by the 
results of autologous bone marrow transplants of SAX vector -infected bone 
marrow cells administered to cynomolgus monkeys. For these experiments, 40-60 
ml of heparinized bone marrow was collected from the animal. Following gel 
sedimentation and Ficoll/Hypaque separation to remove red blood cells, the 
washed bone marrow cells were infected with the SAX vector, either by 
cocultivation with S3A producer cells for 18-24 hours or by incubation with 
S3A-derived cell-free supernatants for 2 hours. Daring the separation and 
infection of marrow cells, the animals underwent total body irradiation, 
receiving a midline dose of 1000 rads. There is no animal model for ADA 
deficiency and, therefore, the monkeys required lethal irradiation in order to 
ablate their remaining marrow and make space for the treated cells. The 
SAX -infected bone marrow cells were then infused back into the same animal. 
Following transplantation, the animals were maintained in gown and glove 
isolation, and received transfusions of blood products, antibiotics and 
[284] Recombinant DNA Research, Volume 12 
