concentration of of 100 ng/ml. Human recombinant interleukin 6 will be added 
to the supernatant to reach a final concentration of 50ng/ml. 
6.6.3 The CD34-enriched bone marrow or peripheral blood cells will be resuspended 
in this transduction media at a concentration of 1 X 10 6 cells/ml. The 
suspension will be transferred to Nunc T175 flasks and transduction media 
added to bring the final concentration of the cells to 1 X 10 5 /ml. 
6.6.4 The flasks will be incubated at 37 degrees C in 5 % CO 2 and 80% humidity for 
20-24 hours. 
6.6.5 The cells and media will be removed from the flasks and pelleted in 50 ml 
conical tubes at 1200 rpm for 5-10 minutes. The pellets will be resuspended 
the same volume of fresh transduction media and transferred back into the 
same T175 flasks. 
6.6.6 After another 24 hours incubation the cells will again be removed from the 
flasks, pelleted, resuspended in fresh transduction media and returned to the 
flasks. 
6.6.7 After a third 24 hour incubation, the cells will be removed from the flasks and 
transferred into 50 ml conical tubes. The flasks will be rinsed once with 20 ml 
PBS each. 5 ml of trypsin (Gibco) will be added to each flask and allowed to 
sit at room temperature for 5 minutes to dislodge cells adherant to the flask. 
The flasks will be rinsed again with 20 ml TCI 99. 
6.6.8 The cells will be pelleted at 1200 rpm for 5-10 minutes and washed twice more 
with heparinized TCI 99. Aliquots of cells will be removed for counting, 
microbial cultures, and other analysis (Section 6.6.9). If no bacterial or fungal 
contamination is found on gram stain and cultures, the thawed, transduced 
cells will be admixed with the thawed, non-transduced bone marrow and 
peripheral blood cells and reinfused into the patient on Day 7 of treatment (see 
Section 6.8). 
6.6.9 After the infection protocol is completed, the CD34+ cells will be evaluated for 
the efficacy of transduction. DNA will be extracted from an aliquot of cells 
and analyzed by PCR for proviral MDR-1 cDNA. Additional aliquots will be 
analyzed by a Rhodamine efflux assay and by immunofluorescence using the 
MRK-16 antibody for MDR-1 gene expression. The clonogenic plating 
efficiency in methylcellulose cultures will be determined as a measure of 
enrichment and cell viability. 
6.7 Cryopreservation of Transduced CD34-Enriched Cells 
Cryopreservation solution will be prepared in a 50 ml conical tube and kept at 
4°C: 9 ml heparinized TC199 media + 3 ml DMSO 
The supernatants will be removed from the pelleted cells and the pellets will be 
pooled in a volume of less than 2.25 ml, and a cell count will be performed. If 
the total cell number is < 100 X 10 6 , the cells will be brought to a total volume 
of 2.25 ml with heparinized TC199. If the total cell number is > 100 X 10 6 , 
the cells will be brought to a volume of 4.5 ml with heparinized TCI 99. 
2.25 ml of cells will be placed into 1 (or 2) labelled 5 ml freezing tubes. 8 ml 
of autologous plasma is added to the cryopreservation solution, and then 2.25 
ml of this mixture is added to each freezing tube. The samples will then be 
frozen in a controlled rate freezer and stored in vapor phase of liquid nitrogen. 
6.8 Reinfusion of Non-transduced and Transduced Cells 
6.8.1 Infusion Instructions: The bone marrow and PBSC will be infused 3 days 
following the last ifosfamide dose by the transplant team. Non-transduced and 
transduced cells will be infused simultaneously. Premedicate patients with 
hydrocortisone lOOmg I VP, acetominophen 650mg PO and diphenhydramine 
25 mg IVP 30 mins before reinfusion. Cells are infused intravenously at the 
bedside over 30 - 120 minutes without a filter after rapid thawing in a 37 
6.7.1 
6.7.2 
6.7.3 
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Recombinant DNA Research, Volume 18 
