3) On the following day, the medium is removed and stored; the cells are 
transferred to a centrifuge tube, pelleted at 500g for 15 minutes, resuspended 
in fresh medium with cytokines, and returned to the dish. 
4) The infection cycle is repeated as in step 2 for a total 4 days. 
5) The growth of the cells is monitored by periodically counting suspended cells 
using a hemacytometer. 
6) The cells are pelleted by centrifugation at 500g for 15 minutes. The pellet 
is then washed twice in cold PBS and resuspended at a cell concentration of 1 
x 10 6 /ml and viably frozen. 
I. MEASUREMENT OF TRANSDUCTION EFFICIENCY AND EXPRESSION OF THE TRANSGENE 
The infected CD34 + cells will be tested for transmission of the human GC gene by the 
R-GC vector using the immunocytochemical method for GC as described in section V.3 and by 
Southern analysis as well as by analysis of GC enzymatic activity in the cell pool (see 
below). Immediately after harvest, the CD34 + cells will be viably frozen according to the 
protocol provided in the appendix for the separation of CD34 + cells. We have experience 
with multiple preparations, that subsequently thawed cells are 80% viable. This 
characteristic permits us to evaluate the efficiency of gene transfer and expression in 
| CD34 + cells during the 3 week interval when the supernatants are being tested for bacterial 
; and viral contamination (described below). For characterization of the efficiency of gene 
transfer, one 10cm plate of CD34 + cells (2xl0 6 ) at the end of infection will be expanded 
for 5 days. This will yield ~10 7 cells. These cells will be assayed for transduction 
efficiency and expression of the transgene. We have selected a minimal transduction 
efficiency of 20% as acceptable because this has resulted in average incremented activity 
of the target cell pool equal to 2-4 times the activity of controls (20-40 times the 
activity of Gaucher patient cells) (see Section IV. E). We will accept the cells if the 
enzymatic activity of the pool is within the range of 100-500% of control cells and 20% of 
the cells stain for the transgene product. 
J. TESTING OF FINAL PRODUCT FOR CONTAMINATION BACTERIAL AND VIRAL CONTAMINATION 
An aliquot of the CD34 + cell mixture at the end of the infection will be tested for 
viability using eosin exclusion. A sample will be submitted to our contract laboratory for 
gram stain and culture for bacterial and fungal contaminants, mycoplasma screen and a 
pyrogenicity test. An evaluation of contamination by replication competent retrovirus (RCR) 
will be made by testing the pooled supernatants from the transduction step. An aliquot of 
100 ml, representing more than 10% of the supernatant, will be amplified on 3T3 cells and 
the resulting supernatant tested in an S+/L- focus assay on PG-4 cells. We will also 
perform a BAG mobilization assay in our own laboratory. Failure to pass any of these tests 
will preclude use of the transduced cells in a patient. 
K. INFUSION OF TRANSDUCED CELLS 
Frozen CD34 + cells will be thawed using a slow dilution method to provide optimum 
viability and recovery of frozen cells. If necessary, concentration of progenitor cells 
will be accomplished using the CEPRATE™ SC Stem Cell Concentrator, as described in the 
appendix. An IND will be obtained for the use of this device in this protocol. The dose 
of CD34 + cells will be 2 x 1 0 6 /kg . 
On the morning of infusion, the following data will be obtained and recorded. 
• Brief medical history and interim clinical status 
• Complete physical examination including temperature, pulse, respiratory rate 
and blood pressure 
• Laboratory studies: CBC, differential, platelet count, BUN, creatinine, SG0T 
SGPT, alkaline phosphatase, bilirubin and urinalysis. Marrow will be thawed 
as outlined and infused through the patient's central venous catheter. 
Recombinant DNA Research, Volume 17 
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