be used in tests of its safety including sterility and tests for the presence RCR. These 
tests will include the BAG mobilization assay done in our own laboratory and the extended 
S+/L- focus assay performed by our contractor in PG-4 feline cells using 5% of the 
production run after amplication in 3T3 cells. 
In an effort to try to evaluate the occurrence of RCR during an extended period of 
culture, we will attempt to extend the culture period to three weeks in a post production 
run. This step should yield another 5 liters of supernatant. We have been successful on 
a pilot scale with the cc-2 producer to achieve this goal. Again, 5% of the post-production 
run will be assayed for RCR by amplication in 3T3 cells and performance of the S+/L- 
extended focus assay in PG-4 cells. 
F. G-CSF PRIMING AND COLLECTION OF A MONONUCLEAR CELL FRACTION 
G-CSF mobilization will be used in patients with Gaucher disease to increase the 
number of PBSC in the blood. Candidates will receive G-CSF at a dosage of 5 mcg/kg/day by 
subcutaneous injection on consecutive afternoons . Injections must be scheduled to begin 
on a Wednesday , in order for leukopheresis and laboratory processing to be performed on 
consecutive weekdays of the following week. Daily morning WBC count and differential counts 
will be obtained, including weekends. Daily monitoring is essential, since WBC can rise 
rapidly on G-CSF. If WBC count is > 45,000/ml, G-CSF dose is to be reduced to 3 mcg/kg/day. 
If WBC count is > 75,000/ml, G-CSF is to be discontinued. 
Leukopheresis procedures will start on day 6 (Monday), to be continued on a daily 
morning schedule until a total mononuclear cell (MNC) yield of 7 x 10 8 /kg is obtained. It 
is anticipated that 3-5 collections (i.e., Monday through Wednesday, or Monday through 
Friday) will be required for the majority of patients to achieve this yield. If a sufficient 
number of MNC is not collected by day 10 (Friday), daily G-CSF may be continued at the 
discretion of the patient's hematologist/oncologist and the Principal Investigator, with 
leukoph3resis resuming on day 13, until an adequate MNC dose is obtained. If cytopenia (WBC 
< 3,000/ul or platelets < 50,000) develops during, or as a result of leukopheresis, the 
procedure will be postponed until recovery. G-CSF will be continued through this interval. 
Seven to ten liters of the patient's blood will be processed, per procedure, depending 
on patient's total blood volume and hematocrit. Samples from each mononuclear cell product 
will be obtained for hemoglobin, hematocrit, total WBC and differential, platelet count; 
colony assays; flow cytometry; and microbiology. Each mononuclear cell product will be 
enriched for CD34 + cells on the day of collection. 
G. ENRICHMENT OF CD34 + CELLS 
The mononuclear cell (MNC) fraction will be enriched using the Ceprate™SC Stem Cell 
Concentrator. A copy of the details of the procedure are included in the appendix. In 
brief, the MNC are labeled with the 12.8 biotinylated anti -CD34 antibody and loaded onto 
a CellPro column which permits separation of the CD34 + cells. Typical enrichment is 40-50 
fold and the cells are usually more than 80% pure by FACS analysis (See appendix). At the 
end of the separation, the CD34 + cells will be viably frozen as described in the protocol. 
H. TRANSDUCTION OF CD34 + CELLS 
The CD34 + cells obtained are transduced according to the procedure outlined below. 
1) CD34 + cells from peripheral blood are suspended in medium containing a mixture 
of cytokines. The mixture consists of rhSCF (lOng/ml), rhIL-6 (lOng/ml), and 
rhil-3 (lOng/ml) in complete LTBMC. The cells are plated into 10 cm dishes at 
an approximate concentration of 2 x 10 6 . The cells are incubated for 6 days. 
2) For infection, the viral supernatant is added to the dish of cells, once or 
twice daily. Protamine sulfate is added to yield a final concentration of 4 
ug/ml . The volume of supe added will be approximately 2 ml with 5 ml of media. 
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Recombinant DNA Research, Volume 17 
