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a long-term culture system to study early murine hematopoietic cells. A number of 
researchers have successfully modified the conditions to permit similar studies on human 
marrow. The results of using such a model system have been encouraging as results 
similar to that seen in colony-forming cells have been seen in long term marrow culture 
initiating cells (14). The GC gene itself can be transferred and expressed in appropriate 
cells as determined in this model system. 
Large animal experience with retroviral mediated gene transfer into marrow cells 
A number of studies have been published on gene transfer into bone marrow cells of 
outbred animals followed by autologous transplantation (reviewed in reference 16). Bone 
marrow stem cells of rhesus monkeys have been successfully transduced by two groups 
(26-28). Multilineage reconstitution of transduced cells was reported and long-term 
expression of the transferred gene obtained (27). Rhesus CD34+ cells have been 
transduced and autologous transplantation with these cells has led to long-term 
reconstitution with Neo R -gene marked cells (28). Similar results have been obtained 
transducing rhesus CD34+ cells with an ADA gene vector (Bodine D, Williams D, 
Donahue R, and Nienhuis A, personal communication). Hematopoietic stem cells from 
dogs (29) and cats (30) have also been transduced successfully. The GC gene has been 
transferred into rhesus hematopoietic cells and gene transfer into CD34+ progenitor cells 
has been very efficient in vitro (2 animals) using a supernatant infection with IL-3, IL-6 
and stem cell factor. Autologous marrow transplantation of rhesus CD34+ cells which 
were transduced with peripheral blood and bone marrow have been followed for up to 
four months (unpublished studies). 
1.4 Clinical Data 
Human experience with retroviral mediated gene transfer into marrow cells 
In order to study the role of autologous marrow in relapses of patients undergoing 
autologous bone marrow transplantation (ABMT) for either acute myelogenous leukemia 
or neuroblastoma, harvested marrow has been marked by RMGT. These ongoing studies 
have not only been fruitful in determining the role of infused autologous marrow in 
contributing to the relapse (31), but also in demonstrating RMGT into hematopoietic 
cells. Patients have now been followed for up to one year after ABMT. Analysis of 
these patients has shown gene transfer into all lineages of marrow-derived cells including 
monocytes, neutrophils, and T and B lymphocytes. Also encouraging is the detection of 
transcribed RNA from the retroviral vector transferred into these cells. The human 
studies, using vectors very similar to those to be used in this study but expressing a 
bacterial antibiotic resistance gene, imply that RMGT into very early hematopoietic 
progenitor cells is possible and that the expression is preserved long-term. Since the 
marrow was not cultured in the presence of growth factors during the transduction 
period, the gene transfer efficiency in these studies may be lower than would be expected 
using the procedures proposed in this protocol. 
Recombinant DNA Research, Volume 17 
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