Human Gene Therapy Subcommittee - 11/21-22/91 
in bone marrow cells. He described experiments in which K562 cells were transduced 
with the LNL6 retroviral vector for 2 hours. One-half of the transduced cells were 
cryopreserved for 24 hours, and the remaining cells remained in culture. Data from 
these experiments indicate a 25-50% reduction in the number of transduced cells when 
cells were cryopreserved. When the transduction time was increased to 4 hours, the 
number of viable transduced cells was reduced even further. 
Dr. Cometta presented in vivo data in which the murine lymphoma cell line, EL4, was 
transduced with LNL6 and injected into mice at the time of transplant. Animals were 
sacrificed at various time points, and cell suspensions of their organs were obtained. 
Neo R positive cells were detected in all of the various cell populations. EL4 cells 
contain a variety of replication-competent retroviruses of murine origin and their in vivo 
effect is unclear. Dr. Parkman asked how many EL4 cells were injected. Dr. Cometta 
said that between 10 6 and 10 7 EL4 cells were injected. Dr. Parkman noted that 10 7 EL4 
cells is enough to kill a mouse; therefore, this model does not imitate a patient with 
minimal disease. Dr. Parkman said that the murine data would be more convincing if 
smaller numbers of EL4 cells had been injected. Dr. Cometta said that they had 
injected animals with 100 cells but that they did not develop tumor. 
Dr. Cometta addressed the issue of cell transduction efficiency. A 6-7% transduction 
efficiency has been observed with the LNL6 retroviral vector; this rate is consistent with 
data obtained from other laboratories using this vector. The colonies grew from only 2 
of 5 patients in methycellulose; 1 patient had ALL, and the other AML. He explained 
the importance of the leukemic colony assay and described attempts to improve the 
growth of cells, such as the addition of stem cell growth factor (SCGF), IL-4, and IL-6. 
These data are preliminary, and no positive results have been obtained. He presented 
data obtained from mixing experiments demonstrating that normal cells can be separated 
from leukemic cells using a fluorescence activated cell sorting (FACS). 
Dr. Cornetta described experiments demonstrating the absence of replication-competent 
helper vims in the LNL6 vector preparations. He explained that the NIH 3T3 
amplifications were performed on short-term cultures obtained from patient blast cell 
preparations. 
Dr. Cometta stated that bone marrow sampling will be performed only at the time of 
transplant. PCR, Western blot analysis, and leukemic colony assays will be performed on 
blood and bone marrow samples obtained from patients in order to monitor cell 
transduction. Dr. Mclvor inquired if the methylcellulose colony assay distinguished 
normal from leukemic cells. Dr. Cornetta responded that normal and leukemic colonies 
could be distinguished morphologically. The leukemic colonies hemoglobinize, creating a 
red appearance. Normal GM colonies are larger than the leukemic colonies. Dr. 
Mclvor asked if the investigators will be using methylcellulose culture in the presence of 
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Recombinant DNA Research, Volume 15 
