Recombinant DNA Advisory Committee - 2/10-11/92 
Dr. Leventhal asked about the volumes used on the selection columns in the clinical 
experiments with normal volunteers and the number of CD34( + ) cells recovered. Dr. 
Dunbar responded that following multiple washes and density separation, cells were 
resuspended at a 1 x 10 8 cells/ml. Other investigators have reported that clinically 
relevant volumes were easily eluted from these columns either from peripheral blood or 
from bone marrow. Dr. Dunbar noted that the other investigators' data were from 
smaller-scale columns, with between 10 8 and 5 x 10 8 total cells. When 5 x 10 8 cells were 
added to a small column, between 2.5 x 10 s and 7.5 x 10 5 cells were eluted in 4 mis 
volumes. One advantage of this system is that there is not a lot of pre-CD34 enrichment 
processing necessary. 
Dr. Gellert asked for clarification concerning the identity of the CD34( + ) stem cells and 
how long their progeny could be expected to survive. Dr. Dunbar responded that the basic 
reason to use this fraction was to have a smaller target population. Based on SCID mouse 
data when highly purified transduced human CD34( + ) cells were administered, human T 
lymphocytes were detected in the thymus out to six months. Therefore, it was 
hypothesized that the stem cell activity is included in CD34( + ) cells though the frequency 
is rather low. Whether or not there will be T lymphoid engraftment from CD34(+) cells 
in humans can only be answered by the kind of experiment being proposed in this 
amended protocol. To date there have been no allogeneic BMT done using CD34 
enriched populations where donor versus recipient cells could be examined. 
Dr. Geiduschek queried as to what gets "torn off' when CD34( + ) cells are eluted from the 
column. Dr. Dunbar responded that the CD34 antigen does not seem to come off the 
cells, but that either the avidin-biotin breaks up or the primary antibody falls off, or both. 
After the cultured cells are examined several hours later, there is little antibody that can 
be observed. After three days, no antibody can be seen bound to the cells. Dr. Dunbar 
confirmed Dr. Geiduschek's conclusion that this result indicates that when the cells are 
reinfused into patients, no foreign antigens are present. 
Dr. Doi had two questions relating to the percent transduction of cells: (1) Was a 
variation found between different people or was it fairly constant? and (2) Could the 
method used to concentrate the target cells be used to concentrate transduced cells and 
measure ADA? Responding to Dr. Doi's first question, Dr. Dunbar stated that there was 
a reasonable amount of variation even among normal volunteers. Between 1 and 35% 
transduction efficiency has been observed in bone marrow using the same conditions 
although the 1% transduction rate may have been a result of problems with the growth 
factors. In peripheral stem cell experiments, between 3 and 10% transduction efficiency 
has been observed. Currently, there is no explanation for the variation; but it is hoped 
that much higher efficiencies can be obtained using G-CSF mobilized cells. In response to 
Dr. Doi's second question. Dr. Dunbar stated that it was conceivable to take the 
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