Recombinant DNA Advisory Committee - 03/1-2/93 
Dr. Miller stated that he has screened the proposed vector. The oncogenes have been 
removed, and the MDR cDNA extends through approximately 50% of the vector. No 
other additional open reading frames were identified. The investigators should describe 
the additional sequences that are at the tail of the vector. Dr. Post inquired about the 
sequences that are upstream from the MDR cDNA. Dr. Miller said that the upstream 
sequences are a derivative of a leukemia virus and contain the packaging signal. Dr. 
Post asked if there was any gag upstream from the cDNA. Dr. Miller responded that no 
gag sequences are present. The vector is approximately 9,000 base pairs (bp) from the 5' 
long terminal repeat (LTR) to the 3' LTR. The MDR cDNA is approximately 4,000 bp. 
Dr. Miller explained that the investigators provided data derived from only 1 experiment 
demonstrating their ability to transduce long-term reconstituting stem cells in mice. This 
experiment was performed by co-cultivation of the marrow cells with the virus producing 
cells. However, the human study will use virus supernatant for transfection as opposed 
to co-cultivation with producer cells. Virus supernatant has been demonstrated to be 
much less efficient than co-cultivation for transferring genes to bone marrow cells. The 
virus titer projected for this vector is 10 4 colony forming units (CFU) per ml. This 
concentration is much less than other investigators have reported as required for 
successful transduction of bone marrow cells. The in vivo transduction data is 
insufficient to support the proposed study. Dr. Parkman said that the investigators 
should perform additional long-term in vitro human experiments with the proposed 
vector. Dr. Miller asked the investigators if there is a large animal model that would 
support this study. 
Presentation-Dr. Bank 
Dr. Bank introduced his collaborators: Drs. Steve Goff and Charles Hesdorffer of 
Columbia University, New York, New York, and Dr. Michael Gottesman of NIH, 
Bethesda, Maryland. Dr. Bank explained that he had reviewed the comments made by 
the RAC in response to Dr. O'Shaugnessy's protocol, and that this protocol addresses all 
of the RAC members recommendations. Dr. Bank said that he has PCR data 
demonstrating that human CD34( + ) cells can be transduced with the proposed vector. 
In response to Dr. Geiduschek's concerns about MDR expression. Dr. Bank explained 
that gene expression will be monitored by fluorescence activated cell sorter (FACS) 
analysis using the 17F9 antibody. When unselected bone marrow cells are transduced 
with the MDR vector, 10-20% of the cells demonstrate a 5- 10-fold increase in MDR 
expression. One CFU-granulocyte/erythrocyte/megakaryocyte/monocyte (GEMM) 
resistant colony assay was performed using CD34( + ) transduced cells in which 2 of 7 
resistant colonies grew. Dr. Bank stated that he is currently in the process of defining 
the endpoints of this study to determine efficacy. 
Recombinant DNA Research, Volume 17 
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