Recombinant DNA Advisory Committee - 09/14-15/92 
clinical protocol involving ABMT for non-myeloid neoplasia. The inclusion criteria are 
limited to patients with non-myeloid neoplasms, e.g., breast carcinoma, to prevent the 
possible mobilization of leukemic cells. These patients have received growth factors that 
will mobilize their peripheral blood progenitor cells. These progenitor cells are then 
harvested and stored for transplantation. Patients participating in the gene marking 
portion of the study, will have 25% of their stored peripheral blood progenitor cells 
transduced with the neo R gene. These transduced cells will be transplanted into the 
patient in addition to the nontransduced cells. Patients will be monitored over time to 
detect the presence of gene marked cells following transplantation to determine if the 
administration marked pluripotent stem cells result in continued expression of the neo R 
gene in both myeloid and lymphoid populations. 
Dr. SchLcning presented preclinical in vivo data obtained using the canine model to 
address the issue of continued gene expression in both myeloid and lymphoid cells. 
Long-term gene expression has been observed out to 3Vi years following transplantation 
of transduced marrow cells in irradiated animals. Two different methods of transduction 
were use* One method used a co-cultivation procedure in which marrow cells were 
cultured with the vector producing cells for 24 hours and subsequently incubated in a 
long-tern marrow culture with vector supernatant for an additional four days. 
Prelimina r y in vitro data suggest that expanding the exposure time to the vector- 
containii supernatant increases transduction efficiency. The animals were pretreated 
with cytc an to enrich for nondifferentiating and stem cells. Cytoxan also stimulates the 
stem cells to cycle which is an important prerequisite for retroviral transduction. 
In recent i vivo experiments, animals underwent bone marrow aspiration and their 
marrow cells were cryopreserved. Following bone marrow harvest, the animals received 
growth factor to stimulate the early progenitor cells. Six days later, the animals were 
leukapherised, and the cells were enriched for Class II antigen positive ( + ) cells which 
have bee.i shown to contain the stem cell fraction in the canine model. These enriched 
cells were cocultivated for 24 hours with vector producing cells followed by continued 
incubation with long-term marrow cells for 1 1 days. These cultures were replaced with 
fresh vector containing media every other day. The animal was then lethally irradiated 
and both transduced and untransduced cells were readministered. Three animals have 
been transplanted to date using a modified protocol that is very similar to the proposed 
human experiments. 1 x 10 7 nontransduced cells and 1 x 10 6 transduced cells were 
transplanted per kilogram. All three of these animals engrafted; one death occurred due 
to infectious complications. The remaining two animals have survived out to 180 days 
post-transplant. Drug resistant colonies from both lymphocytes and marrow cells have 
been obtained out to 22 weeks in these two animals. These data suggest that pluripotent 
stem cells have been transduced in these animals. 
Dr. Schuening presented in vitro human data indicating that 24 hour co-cultivation of 
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Recombinant DNA Research, Volume 16 
