We will document these characteristics for the VPL of R-GC chosen to be used to establish 
the master cell bank. These stability tests will be carried out in the principal 
investigator's laboratory. In addition, the VPL will be evaluated for its ability to 
produce a viral supernatant that can correct the enzyme deficiency of GC in CD34 + cells 
obtained from Gaucher patient bone marrow. We are fortunate to have a large supply of 
patient marrow obtained during a knee replacement. This otherwise discarded material is 
very useful in evaluating vector quality, stability, and determining the optimum conditions 
for infection as described earlier. 
Once we have selected and characterized the best producer line, we will test it for 
contaminating materials according to the guidelines published by the FDA and recommendations 
communicated to the RAC. Because of recent concerns about RCR, we will incorporate 
sensitive tests for the presence of helper virus. We expect this testing to be complete 
by mid summer 1993 due in part to the near term completion of the Human Gene Therapy 
Applications Laboratory (HGTAL) where we will perform some of the testing and production 
of the supernatants. Testing, to be performed by a contract laboratory, will include the 
following to be performed on the master bank (MCB) of the VPL: 
Sterility Cell culture ID 
Isozyme and Cytogenetic Analysis Mycoplasma 
Extended XC Plaque Extended S+/L- Focus 
MAP Antibody Production In-Vivo Virus 
In-Vitro Virus Co-Cultivation with RD Cells 
Bovine Viruses Porcine Parvovirus 
In addition, the supernatant of the MCB will be amplified in 3T3 cells and the 
supernatants exposed to PG-4 (feline) cells and re-tested by the S+/L- focus assay. As a 
measure and an approximation of the clinical application, the MCB will be used to generate 
a viral supernatant with a titer of 10 7 . This supernatant will be used in a study that 
maintains human CD34 + cells in culture for 7 days. The human cells will be exposed to viral 
supernatant produced by the MCB at an MOI of 10 virus particles/CD34 + cell. Repeated 
infections of the cell will take place at a frequency of 1/day for 4 days beginning on day 
3 of the culture. The culture supernatants collected during media refreshments and the 
final supernatant will be pooled and tested using the BAG mobilization assay and 
simultaneously submitted to a contract lab for S+/L- focus assay with the PG-4 cell line 
after amplication in 3T3 cells. Copies of the protocols for the testing done by the 
contract laboratory are enclosed in the appendix. 
E. PRODUCTION AND CHARACTERIZATION OF THE VIRAL SUPERNATANT 
Each patient will require 2 x 10 6 CD34 + cell/kg. This is the dose used to 
; reconstitute the bone marrow in patients who have been myeloablated. The dose needed to 
I result in engraftment in an unprepared recipient is unknown. Studies of syngeneic bone 
i marrow transplants in mice of the opposite sex and autologous transplants in dogs using 
i retrovirally transduced marrow suggest that 1-10% of the donor cells can stably engraft in 
the host animal without preparative myeloabl ation 167,941 . These results provide at least the 
possibility that some niche exists for autologously transplanted bone marrow cells. Our 
| proposed study will evaluate this possibility in the Gaucher patient. We have selected a 
dose of CD34 + cells that has been used to reconstitute the bone marrow in myeloablated 
cancer patients. At this dose of cells and using an MOI of 10/1 with daily exposures for 
! 4 consecutive days, approximately 8 x 10 9 virus particles will be required for each patient 
I in the study. At a titer of 10 7 pfu/ml , 800ml would be sufficient transduce the CD34 + cells 
i from a patient (-1.4 x 10 8 cells). In the preparation of the supernatant, we plan to 
! produce 5 liters from the producer line MCB (See appendix for the protocol on raising 
i supernatants). We will use this production run for testing the titer and confirming the 
transduction efficiency of the lot and its ability to express the GC gene in our stock of 
CD34 + cells obtained from bone marrow of a Gaucher patient. This production run will also 
Recombinant DNA Research, Volume 17 
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