transient circulation of marked cells presumably derived 
from successfully transduced committed progenitors- 46 
Incorporation of the modifications found to increase gene 
transfer efficiency in the murine models has also improved 
the results in primates. 6/6 primates transplanted with 
5FU-primed marrow transduced in co-culture with a high-titer 
but helper-contaminated producer line in the presence of IL3 
and IL6 were positive for proviral DNA in hematopoietic 
tissue at 6-12 months. 42 3/7 animals transplanted with 
CD34-enriched transduced marrow had the marker gene 
detectable in marrow, peripheral blood, purified 
granulocytes and T cells at levels of 1-10% for greater - 
than 100 days post-transplant. 32 However, the presence of 
high-level helper virus contamination of the producer cell 
line used in these experiments, and chronic viremia in these 
animals makes interpretation of these results difficult. 42 
Three of these animals subsequently developed T-cell 
lymphomas related to the helper virus contamination, (see 
Section 3.5) 
Preclinical studies designed to directly assess the 
feasibility and efficiency of gene transfer to human stem 
cells or long-term repopulating cells are not possible. 
Various surrogates have been proposed, including the long- 
term bone marrow culture initiating cell assay (LTBMCIC) . 20 
However, it is not known if this assay really predicts the 
in vivo behavior of human stem cells. Human long-term bone 
marrow culture initiating cells have been successfully 
marked by retroviruses. 47 ' 50 Several different laboratories 
have shown that up to 40% of CFU-C cultured out of 5-8 week 
long-term bone marrow cultures initiated with retrovirally- 
transduced marrow are marked with the proviral genome. All 
of these studies have found that culture for at least three 
days with either hematopoietic growth factors (some 
combination of at least two or three factors, including IL3 , 
with either IL6 , SCF, IL1 or SCF, or autologous stroma are 
necessary for efficient transduction. The transduction 
process had no effect on the number of total long-term bone 
marrow initiating cells present, or on clonagenic progenitor 
production by these cultures. These published studies used 
higher titer viruses than are currently available for 
clinical use. Of note, in all of these studies, the 
transduction efficiency of CFU-C was nearly identical to the 
transduction efficiency of LTBMCIC. 
In our own laboratories we have performed a number of in 
vitro experiments on CD34-enriched bone marrow and 
peripheral blood target cells from normal volunteers and 
patients to assess the efficiency of gene transfer using our 
proposed transduction conditions. This data is summarized 
in Appendix D. Using the proposed transduction conditions, 
10-30% of normal and patient bone marrow CFU-C and 5-50% of 
Recombinant DNA Research, Volume 16 
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