source of malignant relapse following ABMT in malignancies which are 
derived from or involve the bone marrow or the mechanisms of marrow 
regeneration after the procedure (Section 3.3). The availability of transduction 
procedures able to mark harvested progenitor cells means that both issues can 
now be addressed. 
3.2 Origin of Malignant Cells During Relapse 
Retroviral vectors have been successfully used to mark malignant cells and cell 
lines in man. In rodent models, marked malignant cells have re-established 
malignant disease. If residual malignant cells in the harvested marrow could 
be marked, it would be possible to determine whether the malignant cells 
appearing in patients who relapse after ABMT can be derived from the 
infused marrow. Detection of such marked tumor cells would provide 
justification for the continued exploration and development of marrow purging 
technologies, particularly for patients in first remission. This gene marking 
approach appears feasible for neuroblastoma since in vitro studies on a 
number of human neuroblastoma cell lines have shown that these tumor cells 
can be transduced with an efficiency between 5-30% (see Data Appendix), 
while studies of fresh neuroblastoma cells indicate that transduction efficiency 
of clonogenic cells is around 1% (Appendk). 
3.3 Transduction of Marrow Progenitor cells 
Questions concerning stem cell biology and therapy following ABMT can only 
be investigated if the stem cell itself is successfully marked. However, in all 
mammalian species studied, normal stem cells are not in cycle, and none of 
the gene transfer techniques currently available are able to generate stable 
transductants. It is likely that the same will be true for stem cells from normal 
human donors. But stem cell transduction is possible in animals if they are 
pre-exposed to cycle specific cytotoxic agents such as 5-fluoro-uracil. These 
cells can then be effectively transduced with retrovirus vectors, and long term 
gene expression demonstrated in cells capable of self renewal and multi- 
lineage differentiation. 
Because remission marrow for ABMT is harvested from patients during the 
recovery phase from intensive chemotherapy (which usually includes cycle 
specific agents), this marrow too may contain stem cells in cycle. Although 
there is no in vitro assay for the human stem cell we have successfully 
transduced marrow from normal donors, and from patients post chemotherapy 
(AML and neuroblastoma patients). Both show transduction of multilineage 
progenitors (GEMM colonies) and of clonogenic cells in long term marrow 
culture. The efficiency of transduction into clonogenic precursors for both 
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