time to engraftment with a granulocyte count >500/mm3. was 34 
days: one patient failed to engraft due to metastatic marrow disease 
and 3 others died of transplant-related complications at 14. 14 and 
17 davs. two with marrow evidence of early engraftment. The 34 
davs to .engraftment is longer than is genera lly achieve d with use of 
whole marrow as the autograft (21-28 davsl. In the present study. 
patients will be transplanted with > 5x1 QZ buffv coat cells/Kg that 
have not been CD34+ selected nor transduced. This number of buffy 
coat cells is known to be adequate for full marrow reconstitution. 
No greater than 30% of the harvested marrow will be used to 
separate the CD34+ subpopulation and for retroviral transduction. 
These CD34+ cells will be reinfused along with the >5x101 buffv 
coat cells/Kg. Because adequate numbers of un-manipulated buffy 
coat cells will be infused as the autograft, we do not anticipate that 
the addition of the CD34+ cells will decrease the safety and efficacy 
of the autograft. 
In retroviral transduction studies of murine stem cells and 
progenitors, the efficiency of neo gene transfer increases from 
approximately 10% using IL-3 alone in the transduction culture to 
54% in the presence of both IL-3 and IL-6 (Arthur Nienhuis. personal 
communication!. Similar data with human stem cells and 
progenitors is not available comparing IL-3 to IL-3 plus IL-6 to IL -3 
plus IL-6 plus SCF, However, it is known that 10 - 25% of CD34+ 
human cells are marked with the neo gene using the 2-6 dav culture 
duration with the three growth factors (Arthur Nienhuis. Cvnthia 
Dunbar, personal communication!. In the murine system, as the 
efficiency of progenitor transduction increases from 10 to 90%. the 
copy number of neo genes per cell increases linearly to 3-4 copies 
per cell (Arthur Nienhuis. personal communication!. Comparable 
data does not exist for human progenitors. However, only 10 - 25% of 
human progenitors are generally marked with the neo gene under, the 
most optimal conditions currently in use so it is unlikely that this 
subpopulation would have more than 1-4 copies of the neo gene per 
marked cell. As for the efficiency of human stem cell/proaenitor 
transduction increases, however, it is likely the number of neo genes 
per cell will also increase. This could, theoretically, increase the 
risk of insertional mutagenesis. 
Recombinant DNA Research, Volume 16 
[ 101 ] 
