Recombinant DNA Advisory Committee - 09/14-15/92 
murine evidence that peripheral blood derived stem cells reconstitute similar to marrow 
derived stem cells, this result has not been proven in the human system. 
With regard to the differences between LNL6 and LN, Dr. D. Miller will respond to this 
question. In response to the question about the number of animals transplanted to date, 
three dogs have been transplanted. The plan is to transplant a total of six animals in 
order to provide a firm base for the human study. 
Dr. Schuening addressed the earlier question about cost associated with the treatment. 
He explained that the informed consent document has been changed so that the form 
clearly states that patients will not be responsible for the cost of the gene transduction 
procedure. 
The most efficient transduction procedure is obtained by co-cultivation with lethally 
irradiated vector producing cells and subsequent incubation in a long-term marrow 
culture system fed every other day with vector containing supernatant. Earlier 
experiments indicate that 24 hour co-incubation with irradiated producer cells alone 
results in short-term expression of the marker gene. This recent data suggests that long- 
term exposure is necessary for the transduction of committed progenitor cells because 
stem cells replicate very rarely. The long-term exposure increases the likelihood of 
transduction. 
Dr. Post inquired if cell types other than the CD34( + ) selected peripheral blood cells 
are present in the long-term marrow culture system. Dr. Schuening explained that the 
long-term marrow culture system consists of an adherent cell layer that is established 
from the patient's marrow cells at the time of harvest. Dr. Post asked if the adherent 
cells will also be administered to the patient. Dr. Schuening replied that the adherent 
cells also would be returned to the patients. Dr. Post inquired about the potential 
transduction of the adherent cells by the neo R gene. Dr. Schuening said that the 
fibroblasts will probably be transduced by the retroviral vector. Dr. Post was concerned 
that there would be additional stem cells derived from the marrow culture that would be 
transduced in addition to the peripheral blood stem cells. It was unclear how the 
investigators will distinguish between the two cell types. Dr. Schuening answered that 
the adherent layer also will be irradiated; therefore, the marrow culture cells are 
incapable of being transduced. 
Dr. Parkman noted that following co-culture of the progenitor cells with the producer 
cells, the cells will be trypsinized and added to the autologous stroma. In that event, 
murine cells are now mixed with human cells. Although most of the irradiated murine 
cells will die, there exists the possibility that a few mouse cells will remain and be 
administered to the patient. Dr. John Belmont has reported that transduction with 
autologous stroma and cell free supernatants is as efficient as the co-culturing method. 
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Recombinant DNA Research, Volume 16 
