(1300-1500 RPM) at room temperature. A mononuclear cell (MNC) layer was easily visible as 
a white band in the middle of the supernatant. The supernatant above the MNC layer was 
discarded. The MNC layer was transferred to a separate tube for washing the Ficoll from 
the cells. Approximately 20-40 ml of Dulbecco's phosphate buffered saline (D-PBS) (Gibco) 
(lx) was added to the tubes containing the MNC's, or alternatively, KRP-glucose could be 
used. The cells were washed thoroughly at least two times, and the pellets were resuspended 
in culture medium (1640 RPMI (Gibco), 10% FBS, 10% human albumin). The cells were then 
counted and a viability assay was performed using trypan blue exclusion. Cytospin slides 
for Wright stain were prepared and the percentages of resultant isolated M0 and lymphocytes 
were counted. In order to culture macrophages, the mononuclear cells from the preceding 
step were used. The cell count was adjusted to -5 x 10 6 cells/ml with culture medium (1640 
RPMI, 10% FBS, 10% human albumin), and the cells were plated in 35mm culture dishes 
(approximately 10 cm 2 surface) so that the plating density was between 0.2-0. 6 x 10 6 M0/cm 2 . 
The cells were then incubated at 37°C in 5% C0 2 for 60 minutes, and non-adherent cells were 
removed after the incubation. Then 1 ml of D-PBS (lx) was gently placed in the dish, and 
the dish was shaken to remove non-adherent cells left in the bottom of the dish. 2ml of 
! previously warmed culture media was then added to each 35 mm dish, and the cells were then 
incubated at 37°C in 5% C0 2 . The culture medium should be changed only once a week. In 
order to infect human macrophages in culture with MFG-GC, the culture medium from the 
cultured macrophages of the previous step was removed from each culture dish and 1 ml of 
viral supernatant obtained from the amphotropic producer cell line (titer:5 x 10 5 ) was added 
to each culture dish along with Polybrene (8 ug/ml). The dishes were then incubated at 37°C 
in 5% C0 2 for an additional 48-72 hours. The plates were checked to assure an adequate 
number of adherent cells were present. The culture medium containing the viral supernatant 
was removed from each culture dish, and each plate was washed with D-PBS (lx), and the D-PBS 
was removed. Then 1 ml of lysis buffer (0.05M K-P, 1% Triton X-100, pH 6.5) was added to 
each plate, and a rubber policeman was used to detach all cells from the bottoms of each 
dish. The cell lysates were transferred to small Eppendorf tubes and placed on ice. The 
lysates were sonicated and processed for GC enzymatic activity and protein assays as 
described previously. As shown in Figure 10, the enzymatic activity assays of these cells 
demonstrated that the MFG-GC vector is able to impart approximately 75 U/mg cell protein 
to either normal macrophages or the macrophages from patients. This increment of enzyme 
corrects the enzymatic deficiency completely in the cells of patients with Gaucher disease. 
E. GENE TRANSFER AND EXPRESSION IN CD34+ CELLS FROM BLOOD 
CD34 + cells contain a population of pluripotent stem cells capable of reconstituting 
the bone marrow in primates and man. Recent studies by several investigators have shown 
that the human peripheral blood CD34 + cell (PBSC) can be efficiently transduced by 
retroviral vectors. Because of the ability to concentrate these cells in a small volume, 
1 the logistics of ex vivo transduction become greatly simplified. Furthermore, the ratio 
1 of virus to stem cell in infection protocols can be enhanced. This could lead to more 
' efficient transduction. Because of this economy and because the bone marrow of patients 
with Gaucher disease are frequently "packed" (full of cellular material and scar; they 
frequently can not be aspirated), the PBSC are particularly ideal cells for gene 
transfer/therapy studies. The methods used for isolation and flow cytometric analysis of 
CD34 + cells are included in the appendix. The results of a typical >10 fold enrichment with 
87.6% purity are shown in Figure 11. In these laboratory scale columns, enrichment is less 
than in the large clinical scale columns (see appendix). Clonogenic assays reveal an 
i I increase in CFU-GM of about 3 fold (Fi^^effe). 
Results of our initial experiments with CD34 + enriched PB cells from G-CSF primed 
! lymphoma patients show that transduction occurs and leads to expression of the 
g 1 ucocer ebro s i d a s e gene in these cells and their progeny using the MFG-GC retroviral vector. 
In ^fgure ; ;13 are shown enzyme activities of cells harvested shortly after infection in three 
different mixtures of cytokines: 1) IL-3, IL-6, SCF, and GM-CSF; 2) IL-3, I L - 6 , and SCF; 
i 
Recombinant DNA Research, Volume 17 
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