Recombinant DNA Advisory Committee - 2/10-11/92 
RAC could review it directly. Mr. Capron questioned the limited number of animal 
studies with gene marking that had been performed. Finally, there were comments 
regarding the consent form language and patient selection which the investigators had 
modified prior to this RAC meeting. 
Other Comments 
Dr. Mclvor stated that he had two major concerns with this revised protocol: (1) whether 
enriched peripheral blood CD34( + ) cells can be transduced; and (2) can expression of the 
transduced gene be observed in the enriched population following lymphocyte 
differentiation. The RAC needs direct evidence that once the cell population is 
transduced, there will be gene expression. This information has been the RAC's lowest 
common denominator for approval of prior gene therapy protocols. 
Presentation-Dr. Dunbar 
Dr. Dunbar discussed the conditions found to be important for gene transfer to CD34( + ) 
cells. These data were based on extensive experiments performed in Dr. Arthur Nienhuis' 
laboratory over the past ten years, as well as data in murine and rhesus systems from other 
laboratories. The required conditions include: (1) increased viral titer; (2) addition of 
hematopoietic growth factors, such as interleukin-3, interleukin-6, and stem cell factor 
(SCF); (3) treatment of bone marrow donors with 5-fluorouracil prior to harvesting cells 
for transduction; (4) enrichment of the target population for CD34( + ) cells; and (5) co- 
culturing the target population with either viral producing cells or an autologous or 
allogenic stroma. 
The investigators have been constrained to use clinically approved vectors developed for 
titers of l(r to 10 6 infectious particles per milliliter. 5-fluorouracil will not be used in 
patients since it is a chemotherapeutic agent; G-CSF will be used for mobilization since it 
may have the added benefit of allowing cells to cycle. Co-culturing of cells as stated above 
would not be practical because of safety concerns in this human clinical protocol. 
Dr. Dunbar discussed the enrichment of the target cell population. In the past, the 
hematopoietic stem cell has been undetectable. The CD34 antigen is a transmembrane 
protein of unknown function, originally discovered by raising a monoclonal antibody 
against stem cell leukemia. The protein is present on progenitor cells, colony-forming 
cells, and long-term bone marrow culture initiating cells. CD34 selection is an effective 
means of enriching the activity that is desired in this protocol. CD34 selection has been 
performed in some of the rhesus monkey experiments, and these studies have shown that 
CD34( + ) cells could engraft animals and lead to faster reconstitution than if no CD34( + ) 
cells were administered. Human transplantation protocols using CD34 selected bone 
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Recombinant DNA Research, Volume 15 
