delivery of higher dose chemotherapy with less marrow 
suppression . 15,16 
Genetic marking may also help assess the source of relapse 
after autologous transplantation in these three 
malignancies. Contamination of harvested marrow and 
peripheral blood with tumor is clearly inevitable in CML and 
myeloma, and is also likely in metastatic breast cancer. 17 
The utility and feasibility of purging the graft of tumor 
cells remains controversial, and genetic marking studies 
already in progress are designed to address this issue in 
neuroblastoma, acute leukemia and CML. In our transduction 
protocol, there are three potential sources of relapse: 1) 
residual tumor cells surviving conditioning in the patient 
2) tumor cells contaminating the non-transduced 70% of the 
harvested bone marrow and peripheral blood 3) tumor cells 
remaining in the CD34-selected and then transduced marrow 
and peripheral blood cell populations. Whether or not 
marked retrovirally-marked tumor cells can be detected after 
transplantation with a CD34-enriched transduced population 
will be critical information to have before designing 
genetic therapies directed at these diseases. For instance, 
if marked breast cancer cells can be detected at relapse, 
then gene transfer of a chemoprotective MDR1 gene into 
harvested marrow would be contraindicated. 
Three aspects of this proposed genetic marking study differ 
from the autologous hematopoietic transplantation protocols 
previously approved by the Recombinant DNA Advisory 
Committee and already in progress: 1) The use of a CD3 4- 
enriched target bone marrow or peripheral blood stem cell 
population instead of whole bone marrow; 2) The use of a 
longer-term in vitro culture period for transduction (72 
hours versus 6 hours); 3) The inclusion of the hematopoietic 
growth factors IL-3, IL-6 and SCF (stem cell factor) in 
cultures during retroviral transduction. Based on our 
preclinical in vitro and animal studies, we feel that these 
conditions are necessary to achieve a reasonable efficiency 
of gene transfer to progenitors, and to have any chance of 
gene transfer to reconstituting stem cells. 
3 . 2 Enrichment for CD34+ cells : 
Monoclonal antibodies have been developed that recognize a 
115,000 dalton cell surface glycoprotein (CD34) found on the 
surface of 1-3% of human bone marrow cells but not on the 
surface of mature hematopoietic cells such as granulocytes, 
lymphocytes, red cells or platelets. 18 ' 19 Further studies 
have shown that the CD34 antigen is present on virtually all 
hematopoietic progenitors assayed by in vitro colony assays, 
and on more immature long-term bone marrow culture 
Recombinant DNA Research, Volume 16 
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