strongly positive staining. These results indicate that the expression of the transgene 
is independent of the genetic deficiency of the target cell. This fact is both satisfying 
and reassuring that other genetic or epigenetic factors are not relevant to the expression 
of the transgene in the cells of Gaucher patients. We forecast that the detection of 
engraftment will be feasible using GC enzymatic activity as the indicator based on the 
following calculations. 
Starting Specific 
Activity (SA) of 
Gaucher PBL 
Estimated Speci fi c 
Activity of 
Corrected PBL 
(4 X Control ) 
% Genetically 
Corrected PBL 
(% Engraftment) 
Expected SA of PBL 
% Increase 
1 .0 Unit/mg 
40 Units/mg 
0 
1.0 
0 
1 . 0 Unit/mg 
40 Units/mg 
1 
1.39 
30 
1.0 Unit/mg 
40 Units/mg 
5 
2.99 
300 
1 . 0 Uni t/mg 
40 Units/mg 
10 
4.99 
500 
We do not expect a selective advantage of genetically corrected stem cells. However, 
theoretically, enzymatically competent tissue macrophages produced by the engrafted stem 
cells may function to degrade existing glucosylceramide stores. This has been shown in 
patients receiving enzyme therapy. Thus, we plan to measure angiotensin converting enzyme 
and non-tartrate inhibitable acid phosphatase in the plasma of autologously transplanted 
patients. Both of these measures are markers of macrophage storage of lipid and are 
indicators of a therapeutic response. We have shown them to rapidly decline in patients 
treated with enzyme replacement at optimal and sub-optimal doses (Figures 28-2$) . While 
we do not expect Southern analysis to be sensitive enough to detect Tow percentages of 
engraftment, we will perform these analyses as well. Samples will also be preserved for 
semi -quantitative PCR analyses of the human GC in the R-GC vector in subsequent studies. 
If studies of PBL enzymatic activity and indicators of M0 storage do not indicate 
sufficient engraftment, we will proceed to partially ablate the marrow of patients using 
cytoxan alone. The dose will be 7g/m 2 . This dose has been used to stimulate the release 
of progenitors and CD34 + cells to the blood stream by the partial destruction of marrow. 
This stimulates the cycling of the marrow and increases the proportion of stem cells in the 
blood. It reduces the endogenous marrow content and should provide a greater chance for 
the incoming transduced, genetically corrected CD34 + cells to compete with the genetically 
defective bone marrow. This regimen of cytoxan is chosen because it has fewer side effects 
than total body irradiation (TBI) or the typical myeloabl ati ve regimen of higher dose 
cytoxan in combination with busulfan. In more than 20 patients on approved protocols, 
studied by Dr. Ball at the University of Pittsburgh, there have been no mortalities using 
a cytoxan dose of 7g/m 2 . Other centers have successfully employed this regimen of cytoxan 
without mortal ity (92) . In 146 cases reported in the literature, there has been one reported 
mortality. That seriously ill cancer patient died of infection during the period of 
cytopenia. Morbidity related to cytoxan include cytopenia, fever, bleeding, stomatitis and 
gastritis. 
If cytoxan preparation is necessary, it will be done in patients after the results 
on measurements of transduction efficiency and testing for contamination of the infected 
CD34 + cells are complete and the cells released for use. 
Patients undergoing this regimen will be studied for engraftment using the same 
criteria of enzyme activity in PBL and M0 markers as described in detail in this section. 
M. RESPONSE CRITERIA 
The experiment will be judged to be a success by the criteria listed below. 
Group A Patients : Non-Enzyme Treated 
1) An increase in hemoglobin of 2gm/dl 
2) An increase in platelet count of 50% 
Recombinant DNA Research, Volume 17 
[769] 
