marrow culture procedures. The result was probably due to the combination of 5-FU ! 
pretreatment, extended pre-culture of the marrow with cytokines (IL-3, I L - 6 , and stem cell 
factor) in culture, as well as the coculture of the target marrow with a viral producer line 
of related animal origin which may elaborate other stem cell supporting cytokines. 
Sustained, high expression in vivo of the transferred human GC gene resulted from the 
MFG-GC vector. The N2-SV-GC vector by comparison resulted in poor expression in spleen 
colonies and essentially no expression of the gene product in the tissues of long term 
reconstituted animals. This could have been the result of promoter suppression or down 
regulation of the heterologous SV40 promoter. We have not examined the reasons for this 
failure in detail because of the success of the MFG-GC vector. However, the difference is 
not only simplification of the vector. Several laboratories have observed that simplified 
vectors carrying the human GC gene failed to result in sustained expression of the gene in 
vivo. While expression in vitro or short term in vivo could be achieved, expression of the 
GC gene was rapidly lost in vivo. In a recent report by Correll et al , an LN vector 
derivative was successful in achieving sustained expression of the GC gene in vivo. The 
reasons for these differences are not clear especially since it is now known that the MFG 
vector is not greatly different from other retroviral vectors. 
Demonstration of efficient transduction and high expression of the transferred human 
GC gene in the monocyte/macrophage lineage demonstrated in these studies is important to 
the consideration of gene therapy for GD. As discussed earlier, the macrophage is a primary 
target for the therapy of GD. The results provided here demonstrate that macrophage progeny 
derived from transduced HSC repopulate bone marrow, liver, spleen, and lung. These are the 
organs primarily involved in the pathology of the most common phenotype of GD. This is best 
appreciated by our results in liver. The copy number of the human GC gene in liver of 
0.1/genome is congruous with the proportion of bone marrow derived macrophages (Kupffer 
cells) comprising the liver (-15%). The enzymatic activity in the livers of reconstituted 
animals was nearly twice the background activity indicating that macrophages carrying and 
expressing the human gene repopulated the organ. Direct studies of macrophages cultured 
from the animals demonstrated that cells of this lineage carried and efficiently expressed 
the human gene. 
Although it is satisfying to have overcome the problem of low expression, possible 
problems related to overexpression of the gene product in cells might be a cause for concern 
when considering the potential application of gene therapy for GD. However, several lines 
of evidence suggest that the increase in GC expression measured in this study should not 
be problematic. First, the animals in these experiments appeared to be normal for up to 
8 months. Animals receiving secondary transplants continued to express the human GC gene 
for over a year and appeared to be healthy. Furthermore, GC is a consti tuitively expressed 
house keeping enzyme that is not highly regulated. Some variation in its activity occurs 
normally and should be tolerated. In addition, in animal studies of enzyme replacement 
using macrophage targeted enzyme, the glucocerebrosidase activity of Kupffer cells was 
increased by more than 10 fold without harmful side effects'. Moreover, chronic 
administration of 10-100mg of enzyme twice a month to more than seven hundred patients for 
periods of up to eight years has not resulted in any significant clinical problems. 
D. TRANSDUCTION AND EXPRESSION OF THE HUMAN GC GENE IN NORMAL HUMAN AND GAUCHER 
PATIENT MACROPHAGES (M0) 
Human macrophages were cultured from peripheral blood from normal volunteers and 
patients with Gaucher disease following an approved protocol (IRB #910505). Blood was 
collected in 50 cc syringes containing 2.5 ml of Na heparin (1000 /ml). 5% Dextran was then 
added in saline solution in amount that was approximately 10% of the blood volume to a 50ml 
syringe, 5 ml of Dextran was added). The resulting composition was then mixed thoroughly 
and left undisturbed at room temperature for 40 minutes until a clean separation line was 
visible between the supernatant. The supernatant was layered onto Ficol 1 -Hypague 
(Histopague® 1077, Sigma) (F-H). The tubes were then centrifuged for 20 minutes at 530g 
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Recombinant DNA Research, Volume 17 
