Human Gene Therapy Subcommittee - 11/30/90 
Dr. Mclvor said that the issue of transduction of marrow cells required further 
consideration as to vector safety. If enough cells were transduced, eventually one 
would see an insertional oncogene activation event, and this eventuality should be 
considered. The investigators should describe how large-scale transduction, 10^ cells, 
bone marrow cells would be conducted. 
Dr. Mclvor said he was not clear on the follow-up assays that would be performed 
and if the sensitivity of Southern blot analysis and PCR are sufficient to allow 
meaningful analysis. 
Dr. Mclvor said he would prefer to see this study conducted in animals in exactly the 
same fashion as is being proposed in humans before approving the protocol. Animal 
data would allow more confidence to be placed on whether it can be accomplished 
in humans. 
Dr. Parkman said it was important to know the quantity of leukemic progenitor cells 
that contribute to relapse in a patient, and the relative contribution of the marrow to 
relapse as opposed to the host. Current methods of analysis, including PCR, are not 
sensitive enough to allow such detailed quantitation. It is crucial to be able to 
identify each cell that has the neomycin resistance gene in order to perform such 
analyses. 
Dr. Mahoney asked what type of analysis could be used to observe the single cells 
and identify those marked with the neomycin resistance gene. Dr. Parkman said that 
in situ hybridization could be used to establish the ratio of cells containing the 
neomycin resistance gene to the total cell pool. This technique could be used to 
quantitate the marked cells, to differentiate leukemia cells from normal cells, and to 
quantitate leukemic cells in bone marrow. 
Dr. Epstein expressed concern that if this method was employed, and the assay was 
100% efficient in identifying the cells, 90% of the entire cell population would be 
unaccounted for if the maximum efficiency of detecting labelled cells was 10%. 
Dr. Parkman agreed and said his comments were based on the presumption that 
100% of the leukemic progenitor cells would be labeled. This method is a major 
question in the experimental design. The investigators did not provide data 
demonstrating 100% labelling can be accomplished. 
Dr. Mclvor asked if it was possible to sort bone marrow cells on the basis of surface 
markers and character and perform molecular analysis rather than depend on 
morphological studies. Dr. Parkman said that sorting was not possible for Acute 
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