Recombinant DNA Advisory Committee - 2/10-11/92 
Data were shown on marking normal marrow cells; consistent results were obtained from 
two different laboratories. Dr. Cometta achieved 6 to 7% transduction efficiency, which 
compares favorably with other laboratories. A difference was observed between marked 
normal marrow and marked leukemic cells. Leukemic cell colonies in the methylcellulose 
assay tend to vary from patient to patient and have a smaller number of cells. When the 
colonies were plucked and examined by light microscopy, the leukemic cells could be easily 
identified. Also, the leukemic colonies will be assayed for the neomycin resistance gene 
using beta globin as a control. Another assay will be conducted to show that leukemic 
cells are truly marked and not representative of marked marrow. This method will use 
flow cytometry and cell sorting. Preliminary mixing experiments were performed using 
gene marked K562 cells at 1% with normal marrow cells. Following cell sorting, LNL6 
vector was identified by PCR and Southern blotting. The 3T3 amplification assay, which 
detects replication-competent amphotropic virus, appears to be the most sensitive 
biological assay. In addition to biological assays, Western blotting will be performed to 
detect evidence of the viral envelope antigens. 
Dr. Cometta addressed cell cryopreservation. When K562 cells were frozen for five 
minutes, there was a significant rate of transduction. The transduction rate reached a 
plateau at approximately one hour and then leveled off. When K562 cells were frozen for 
two hours, there was a decrease in the growth rate; about half of the expected number of 
cells resulted. Also, colony assay was performed on K562 cells that were frozen for 2 
hours. This experiment yielded only 26% of the expected number of resistant cells. When 
the transduction period was extended to four hours, the growth rate showed 67% of the 
expected number of cells. The colony assay showed a 70% reduction in the number of 
G418 resistant cells. Cryopreservation experiments demonstrated that transduction 
efficiency would still be above the 1% level, which would provide very significant results if 
malignant cells were present in the transplanted marrow. This experiment was repeated 
using leukemic cells taken from two patients who had circulating peripheral blasts and who 
had not undergone chemotherapy. The cells were transduced; no differences were 
observed in transduction efficiencies of these cells. This result suggested that K562 cells 
may be more sensitive to freezing than primary leukemic cells taken from patients. 
Dr. Cometta stated that all of the suggested changes to the informed consent document 
were incorporated except for the references to autologous BMT and harvest. The patients 
will be introduced to this protocol only after they have agreed to undergo autologous 
BMT. The gene marking protocol might influence the patients' decisions regarding 
treatment. The investigators will explain the informed consent regarding autologous BMT 
with the patient, and the patient must agree before he/she will be asked to participate in 
gene marking. This separate informed consent document is for autologous BMT that 
thoroughly explains all the issues of harvest. Patients must sign another informed consent 
prior to the bone marrow harvest surgery. 
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Recombinant DNA Research, Volume 15 
