Stopping Rule 
If at any time during the study, the presence of helper virus is detected, the study 
will be halted until all materials are certified to be helper free. Transplanted patients 
will be monitored for retroviremia with helper virus by testing the serum and white blood 
cells from 50ml of blood by the BAG assay. This monitoring will be repeated every 30 days. 
If there is evidence of retrovi remi a, the patient will be treated with antibodies to helper 
virus. The study will be halted until the source of the retroviremia is determined and 
corrected. If other studies of gene transfer using retroviral vectors indicate a reason 
for stopping this study, this will be done. 
Post Mortem Examination 
It is very unlikely that a patient will die during their participation in this 
protocol. Should death occur, a post mortem examination will be requested. 
C. DESCRIPTION AND CHARACTERIZATION OF THE VECTOR FOR GENE TRANSFER 
The MFG-GC vector was used for the preclinical studies described in Sections IV: C,D 
and E. This vector contains 1035 bases of the gag gene in an open reading frame. We have 
concluded that the MFG vector could be improved for human trials by eliminating the in frame 
gag sequences, thus rendering the vector unable to synthesize truncated gag-related peptides 
of any significant size as well as decreasing the possibility of replication competent 
retrovirus (RCR). We accomplished this by performing partial Smal digestion of the vector 
containing the GC cDNA and inserting an eight base pair Sac II linker 3' to Hae III site 
at the ATG of the half-gag gene in the retroviral sequence. This creates an additional 
mutation in the vector and destroys the open reading frame for the gag polypeptide sequence. 
We confirmed the insertion of the Sac II linker by sequence analysis across the region of 
the Sma-1 site. This sequencing was duplicated by Lark Sequencing Technologies who obtained 
identical results. The sequence analysis is enclosed on disks labeled BA123.SEQ and shown 
in Figure 23. 
The new vector, called R-GC, was co-transfected to the GP+E86 packaging line. Stable 
clones were isolated and screened for the activity of glucocerebrosidase. We have found 
this identification process very useful in selecting clones to be carried on to high titer 
viral producing lines (VPL). The GP+E86 transfected cell line was used to generate a viral 
supernatant. This supernatant was used to infect the psi-crip and GP+envAM12 packaging 
lines. The activity of the human enzyme in these cells is shown in Figure 24. The 
significant increases in the activity of human GC in cells transduced by R-GC demonstrates 
the ability of the vector to transmit the human GC gene and express the gene product. 
Studies are in progress to select clones of the best VPL's which will be compared to MFG-GC 
producers in parallel studies of titer. Transduction efficiency and expression in human 
Gaucher CD34 + cells will be compared to results with MFG-GC. The best VPL will be 
1 characterized and studied for the presence of contaminating materials, especially helper 
| virus. This will be discussed in detail in the following paragraphs. 
D. PRODUCTION AND CHARACTERIZATION OF THE VIRAL PRODUCER LINES 
The following methods were used to develop the MFG-GC viral producer lines. In the 
initial studies with ecotropic producers of MFG, we made comparisons between the MFG-GC and 
N2-SV-GC vectors. The MFG-GC construct was co-transfected with pSV 2 Neo to the ecotropic 
packaging line, Psi-cre, using calcium phosphate precipitation. The molecular ratio of MFG- 
GC and pSV 2 Neo was 20:1. After G418 selection (400ug/ml), 30 clones were isolated and grown 
! up to near confluency in 100 mm dishes. The culture medium of these clones was used as the 
i virus source. As a target cell for titering the virus NIH 3T3 cells were plated at a 
| density of lxlO 5 cells per well in six-well dishes. Polybrene was added to the medium at 
a concentration of 8 ug/ml to facilitate uptake of the virus by cells. After 2 hour 
! incubation at 37°C, the virus-containing medium was removed and new medium was added. 
: Following 48 hours incubation at 37°C, the cells were harvested and assayed for enzymatic 
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