Recombinant DNA Advisory Committee - 09/14-15/92 
marrow cells with vector producing cells results in 5-24% neo R colonies. This data 
corresponds to the information obtained from the canine model. In addition, peripheral 
blood progenitor cells were obtained from a patient participating in the ongoing 
therapeutic trial in which the patient received G-CSF for the mobilization of progenitor 
cells. Following mobilization, CD34( + ) cells were selected. This selected fraction of 
cells was then transduced by 24 hour co-cultivation with vector producing cells. 
Fractionation increases the efficiency of transduction because the number of target cells 
has been greatly reduced. 
The goal of the protocol is to determine if genetically marked peripheral blood 
repopulating cells contribute to long-term hematopoietic reconstitution after autologous 
marrow transplantation, and specifically, to monitor the quantitative differences in long- 
term contribution by marked peripheral blood repopulating cells based on the particular 
growth factor administered prior to bone marrow harvest. One protocol uses interleukin 
(IL) 3 and the other uses G-CSF for mobilization of early progenitor cells. Important 
information will be derived from this protocol regarding which growth factor is the best 
choice for mobilization in ABMT. 
Dr. Schuening described the human protocol. Twenty days prior to transplantation, the 
patient's marrow will be harvested and cryopreserved. Subsequently, the patient 
undergoes cytokine treatment for the mobilization of peripheral blood progenitor cells. 
Four days following cytokine treatment, the patient is leukapheresed and the cells are 
enriched for CD34( + ) cells. Twenty-five of these CD34( + ) cells will then be transduced 
using the 24 hour co-cultivation with producer cells followed by long-term marrow 
culture. Vector containing supernatant will be replaced in these long-term cultures every 
other day in order to extend the exposure time to the retroviral vector thus, increasing 
the transduction efficiency. The LN vector is safer than the LNL6 vector because the 
env sequences have been eliminated, reducing the possibility of homologous 
recombination leading to helper virus production. However, there have been no 
instances of helper virus production with the LNL6 vector to date. If the RAC decides 
that insufficient safety data has been submitted with the LN vector, then the LNL6 
vector will be used. Following the preparatory regimen, the patient will receive the 
transduced and untransduced marrow cells in addition to the transduced CD34( + ) cells. 
After hematopoietic recovery, marrow and peripheral blood cells will be assayed by 
polymerase chain reaction (PCR) for the presence of the neo R gene as well as the 
development of neo R colonies. 
In response to Dr. Walters question regarding the fate of the marked peripheral blood 
repopulating cells, Dr. Schuening responded that based on the in vivo canine model, data 
suggests that peripheral blood derived stem cells behave similarly to bone marrow 
derived stem cells. Following infusion, peripheral blood repopulating cells will migrate 
to the bone marrow which will lead to hematopoietic recovery. Despite the in vivo 
Recombinant DNA Research, Volume 16 
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