to intepret quantitatively or statistically. In the 
murine model system, it is possible to transduce 20% of 
long-term repopulating cells using optimum 
conditions. 10 It is estimated that there is one stem 
cell in every 10 5 murine mononuclear bone marrow 
cells. S8 If this ratio of total bone marrow cells to 
stem cells applies to humans, than we will be 
transducing an average of 1-10 X 10 4 stem cells from 
each patient. Even with a transduction efficiency of 
1% or lower, it would be likely that we would be able 
to detect stem cell transduction in each patient if was 
actually occurring in vitro under these transduction- 
conditions . 
Any evidence of stem cell transfer would encourage us 
to optimize conditions for marrow or peripheral stem 
cell collection, transduction, or transplantation to 
improve the percentage of transduced stem cells. If 
there is no evidence of gene transfer to long-term 
reconstituting cells after transducing cells from 6-10 
patients under the proposed conditions, then we will 
propose new conditions based on ongoing primate and in 
vitro studies. It is likely that better combinations of 
hematopoeitic growth factors able to cycle stem cells 
will become available. We would also consider 
transducing CD34+ cells on either an autologous or an 
immortalized stromal layer. 50 
10.3 Biology of reconstitution following autologous 
transplantation of bone marrow or peripheral blood stem 
cells : As is true in all genetic marking protocols or 
gene therapy protocols, very little can be learned if 
marking is completely unsuccessful, and no transduced 
cells can be identified either short or long term after 
transplantation and engrafment. However, the detection 
of any marked cells long-term will increase our 
knowledge of the biology of autologous reconstitution, 
as discussed in detail in section 3.0. 
10.4 Source of relapse follwoing autologous transplantation 
for malignancy : If any patients relapse with marked 
tumor cells, then analysis would be informative and 
relevant to designing purging regimens for autologous 
transplantation in these three diseases. If 'analysis of 
tumor cell population DNA shows both marked and 
unmarked cells, and insertion site analysis via 
Southern blotting shows a polyclonal pattern, it would 
imply that transduced CD3 4— enriched bone marrow and/or 
peripheral blood contains many cells capable of 
contributing to relapse, and that relapse is often 
polyclonal. If only rare patients relapse with marked 
tumor cells, it would be less informative, but would 
Recombinant DNA Research, Volume 16 
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