long-term repopulating hematopoietic cells 
Four dogs have been transplanted with peripheral blood and/or marrow cells transduced 
with retrovirus vectors containing the human glucocerebrosidase gene. Animals were 
treated with c-kit ligand for 7 days to mobilize peripheral blood repopulating cells and to 
stimulate early marrow progenitor cells to divide. On days 5 and 6 of growth factor 
treatment, dogs underwent leukapheresis. Peripheral blood cells were incubated for 7 
days in a vector-containing long-term marrow culture system which was fed daily with 
retrovirus-containing medium. On day 7 of growth factor treatment, 5 days before 
transplantation, marrow was aspirated from all four legs. Cells were transduced either by 
24-hour cocultivation on vector-producing packaging cells followed by 4-day incubation in 
a vector-containing long-term marrow culture system or by 5-day vector-containing long- 
term marrow culture only. After myeloablative treatment with either 9.2 Gy TBI or 40 
mg/kg of cyclophosphamide, dogs received between 0.12 and 0.26 x 10 8 /kg transduced 
peripheral blood cells and/or between 0.93 and 1.2 x 10 8 /kg transduced marrow cells. 
All four dogs engrafted. After hematopoietic reconstitution, peripheral blood 
granulocytes and lymphocytes were analyzed by PCR for the presence of the human 
glucocerebrosidase gene. The gene has been found up to 18 weeks after transplantation 
in both myeloid and lymphoid cells to date. This suggests transduction of pluripotent 
long-term repopulating cells (Figure 9, Table 6). However, GC enzyme activity in 
peripheral blood cells was not increased as compared to activity in cells from control 
dogs. No obvious difference was found up to now regarding the efficiency of the 
different transduction methods used. 
F. Retrovirus-mediated neo gene transduction into human G-CSF mobilized peripheral 
blood-derived CFU-GM 
Eight patients were treated with G-CSF (16 pg/kg/day) for 7 days and underwent 
leukapheresis daily for 4 days starting on day 5 of growth factor treatment. In seven 
patients an aliquot of the peripheral blood cells collected was then selected for CD34- 
positive cells by avidin-biotin immunoadsorption and transduced by 24-hour cocultivation 
on PA317/LN packaging cells or by 5-day exposure to vector containing supernatant in a 
LTMC system. Between 10 and 51% G418-resistant CFU-GM colonies were observed 
(Table 7), similar to the results obtained with human marrow-derived CFU-GM (Table 
8). This experiment suggests that human peripheral blood-derived CFU-GM can be 
transduced by retrovirus vectors similar to human marrow-derived CFU-GM. 
Transduction of CD34-positive peripheral blood cells appears to result in a higher 
transduction rate as compared to unselected cells. 
G. Retrovirus-mediated transduction of the human glucocerebrosidase gene into human 
hematopoietic progenitor cells 
Several investigators have demonstrated efficient transduction of the human 
glucocerebrosidase cDNA into a substantial fraction of human hematopoietic progenitor 
cells obtained from otherwise normal donors and from patients with Gaucher’s disease 
(19-21). Between 35 and 45% transduced colony forming units (CFU) were observed 
when transducing marrow obtained from patients with Gaucher’s disease. Enzyme 
activity of transduced Gaucher progenitor cells increased 3.5 to 6.2 fold as compared to 
progenitor cells transduced with a vector containing only the neo gene. Transduction of 
G-CSF mobilized CD34 positive peripheral blood cells obtained from normal donors with 
a GC vector resulted in a 1.8 fold increase in GC activity above endogenous levels. 
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Recombinant DNA Research, Volume 18 
