peripheral blood granulocytes and peripheral blood and lymph node lymphocytes 
containing the neo gene is 0.1-10%, which is consistent with the percentage of G418- 
resistant CFU-GM observed. Peripheral blood samples of the dogs are free of helper 
virus and no long-term side effects from the transduction of the retrovirus have been 
observed so far with observation times between 2 and 4 years. One dog (C874) died due 
to metastatic prostate carcinoma 3 years after transplantation. DNA of multiple tumor 
samples was analyzed by PCR for the presence of retrovirus vector DNA. None of the 
samples contained vector DNA (Figures 4a and b), suggesting that the tumor developed 
independent of gene transduction, probably as a late side effect of total body irradiation 
(see detailed description on page 9). Intermittent gene expression is probably due to the 
small number of repopulating cells transduced, which produce committed progenitor cells 
intermittently for limited periods of time. These data demonstrate successful 
transduction of long-term repopulating n.arrow stem cells in a large random bred adult 
animal model without any long-term side effects. 
D. Retrovirus-mediated neo gene transduction into canine long-term repopulating peripheral 
blood cells 
Three dogs have been transplanted with transduced peripheral blood cells and 
untransduced marrow cells after 9.2 Gy TBI. After aspiration and cryopreservation of 
marrow, animals were treated with c-kit ligand for 8 days to mobilize peripheral blood 
repopulating cells. On day 7 of growth factor treatment, dogs underwent leukapheresis. 
Peripheral blood cells were selected for MHC class II antigen-positive cells by avidin- 
biotin immunoadsorption to enrich for early hematopoietic progenitor cells. Class II 
MHC-positive cells were then transduced by 24-hour cocultivation on PA317/LN 
packaging cells and 1 1-day incubation in a vector-containing long-term marrow culture 
system which was fed every other day with new vector-containing medium. After 
otherwise lethal irradiation with 9.2 Gy TBI, the dogs received between 0.05 and 0.12 x 
10 8 /kg transduced peripheral blood cells and, additionally, between 1.8 and 4.1 x 10 8 /kg 
untransduced autologous bone marrow cells. The transduction rate of peripheral blood 
progenitor cells at the time of transplantation ranged from 2% to 18% as determined by 
G418-resistant CFU-GM colonies. All three dogs engrafted; however, one dog (D425) 
died from chronic canine distemper sclerosing encephalitis on day 84 after 
transplantation (see detailed description on page 9). Autopsy was performed and tissues 
from different organs were analyzed for neo-containing sequences by PCR. The results 
are shown in Figure 5. Neo was detected in peripheral blood granulocytes and bone 
marrow; all other tissues analyzed were negative. After hematopoietic reconstitution, 
marrow and peripheral blood cells were examined in the other two dogs for the presence 
and expression of the neo gene. So far, marrows have shown between 1 and 10% G418- 
resistant CFU-GM colonies for up to 48 weeks after transplantation (Table 4). The 
presence of the neo gene in marrow, peripheral blood granulocytes and lymphocytes 
continues to be demonstrated by PCR for up to 60 weeks after transplant (Figures 6-8 
and Table 5). These data suggest transduction of peripheral blood-derived long-term 
repopulating cells. The two surviving dogs remained healthy and free of any side effects 
of the transduction procedure. Both dogs have been repeatedly tested for helper virus 
and none has been detected. 
E. Retrovirus-mediated transduction of the human glucocerebrosidase gene into canine 
Recombinant DNA Research, Volume 18 
[665] 
