transduction into the harvested marrow of patients who are to receive ABMT 
offers the only means of investigating this question for the majority of AML 
patients who lack chromosomal markers. 
3.4 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-Fluorouracil. During 
the recovery phase from this drug there is multi-lineage hemopoietic 
proliferation, and entry of stem cells into cycle. It is not clear if entry into 
cycle is the critical event, but nonetheless stem cells so treated 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. We propose 
to determine if marrow from these patients transduced by the LNL6 vector 
will produce cells containing the marker after ABMT in cells derived from 
multiple different lineages. Should the marker be present at the same insertion 
site in multiple different lineages (see below) this would be good evidence for 
transduction of a pluripotent progenitor, while long term of this marker in the 
same site would be evidence for transduction of a self renewing "stem cell". 
However, the in vitro data presented in the appendix ("Transduction of Long 
Lived Progenitors" suggest that while transduction of relatively long-lived 
committed progenitors occurs, there is no evidence for transduction of a 
multipotent self renewing cell. But even if expression of transduced genes is 
detected only in relatively long lived committed progenitors, we will still be 
able to analyze those exogenous and endogenous stimuli including infection, 
further chemotherapy and recombinant growth factors, which promote 
proliferation of the engrafted marrow. 
Ultimately this information would allow protocols to be devised which would 
induce optimum (stem) cell cycling and responsiveness of marrow pre ABMT 
for optimum recovery post ABMT. The information would also facilitate 
techniques aimed at growing the marrow ex vivo from small aliquots, 
[510] 
Recombinant DNA Research, Volume 14 
