Similar results were obtained in our laboratory when transducing G-CSF mobilized 
peripheral blood progenitor cells from normal donors (Figure 10). GC enzyme activity 
of cells transduced with GC containing vectors was increased 1.3 to 4.9 fold above 
endogenous enzyme activity of control cells transduced with neo containing vectors. 
Enrichment for CD34+ cells increased transduction efficiency 1.2 to 2.7 fold as 
compared to unselected controls. 
CFU-GM colonies, obtained from normal peripheral blood cells which were transduced 
with GC containing vectors, were analyzed by PCR for the presence of the vector- 
derived GC cDNA. Three, five or ten colonies were picked from agar cultures and 
submitted to PCR amplification. PCR primers used were specific for a cDNA-fragment 
of the LTR-GC gene region of the vector and did not amplify the endogenous human 
GC gene in untransduced control colonies. Positive results in at least one of three 
colonies picked from the agar cultures indicate a transduction efficiency of at least 33% 
(Figure 11). 
H. Ex vivo neo gene transduction into human marrow cells followed bv autologous marrow 
transplantation 
Brenner et al has transduced the neo gene into marrow cells harvested from children 
with acute myeloid leukemia in remission who were undergoing autologous marrow 
transplantation (22). The aim of this study was to determine whether relapse occurring 
after autologous marrow transplantation is due to residual leukemic cells in the 
transplant or only to residual disease in the patient. Patients have been followed so faij 
for up to one year after autologous transplant. Two patients have relapsed and in both 
the blast cells contained the neo gene indicating that remission marrow can contribute to 
leukemic relapse. The second important result of these studies is the demonstration of 
the persistence of the neo gene in all hematopoietic lineages for so far up to one year 
after transplantation. These data strongly suggest that it is possible to transduce human 
long-term repopulating hematopoietic cells using retrovirus vectors. 
I. Engraftment of hematopoietic repopulating cells in nonmveloablated recipients 
Using mouse or dog models, investigators have been able to demonstrate engraftment of 
long-term repopulating cells without prior myeloablative treatment of the recipient (23- 
27). Quesenberry et al (26) has shown long-term engraftment (up to 25 months 
posttransplant) of normal male donor marrow transfused into noncytoablated female 
mice when transplanting large numbers of donor marrow (40 x 10 6 cells daily i.v. for 5 
consecutive days, which is the equivalent of marrow from 2 tibias and 2 femurs daily). 
When injecting 40 x 10 6 marrow cells once or 5 x 10 6 cells daily for 5 days, no 
engraftment was detected. Carter et al. (27) transplanted genetically {neo gene) marked 
canine long-term marrow culture cells into autologous recipients with (four dogs) or 
without (three dogs) myeloablative conditioning. Long-term persistence (up to 2 years 
posttransplant) of the neo-marked marrow cells was obser/ed. There was no difference 
in the percentage of neo - marked cells present in dogs that received marrow ablative 
conditioning as compared to those receiving no conditioning. These data suggest that 
successful syngeneic or autologous transplantation of marrow cells is possible without the 
need to create space or open niches for the transplanted marrow by preceding 
cytoreductive treatment. Based on these experiments it is reasonable to expect 
Recombinant DNA Research, Volume 18 
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