Human Gene Therapy Subcommittee - 4/5/91 
Dr. Deisseroth turned to the issue of in situ hybridization, noting the degree to which 
people are interested in solving this problem. Fluorescent probes could be used to 
detect single copy genes in situ; the cells could be scored on a microscope slide for the 
presence or absence of leukemic markers and the presence or absence of the viral trans 
genome. He showed an example of this capabihty in which his group uses fluorescent 
probes that can detect the number of chromosomes or specific single copy genes on 
chromosomes, through direct chemical conjugation of the DNA probes with the 
fluorescent molecules. 
Dr. Deisseroth noted that CML disease was chosen because the leukemic blast crisis 
cells are cells which are in cycle, and their cycling properties are much more active than 
the chronic phase cells that were first used as the model system. The estimates given for 
marking chronic phase cells are probably underestimates, while the marking frequency 
for blast crisis cells will be much higher. Therefore, the model chosen has really skewed 
the situation in favor of being able to detect viral marking and to detect relapse in a 
reasonable period of time in a reasonable number of patients. 
Dr. Parkman indicated that a relapse six months after the marrow transplant would not 
be derived from blast cells which were marked at the time of transplant. Rather the 
relapse is likely to be caused by cells that are more immature at the time of transplant 
and differentiate into blast cells at a later date. When the blast crisis cells may not be 
the cells of origin for the relapse, there needs to be evidence that one can mark the cell 
that will give rise in six months to the blast crisis cells. 
Dr. Deisseroth felt the question was central to the issue of gene therapy. The issue of 
how to insert a virus into the normal human hematopoietic progenitor cell in order to 
produce after transplantation, or after infusion of these cells, a marrow that is dominated 
by cells which possess the introduced genetic information, is critical. In using the CML 
model, the long-term strategy is to first establish whether the marrow is the origin of 
relapse, and then to fractionate the marrow to try to get rid of the cells responsible. 
One can follow through lineage distribution of the retroviral genome the fraction of 
enrichment. 
Dr. Epstein said the Philadelphia chromosome appears in other lineages besides the 
leukemia lineage. When a patient who is in acute blast crisis is treated, what happens to 
all those other lineages that are still Philadelphia chromosome positive? Dr. Deisseroth 
said if the treatment for the blast crisis results in a disappearance of the blast crisis cells 
and a disappearance of all cytogenetically detectable evidence of the leukemia, then the 
leukemia disappears from the other lineages as well. Lineage involvement is removed if 
a cytogenetic remission is induced. Dr. Epstein pointed out that the other lineages are 
not necessarily leukemic, but just carrying the marker. Dr. Deisseroth said if the other 
lineages carry the marker, then they must be leukemic. 
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Recombinant DNA Research, Volume 14 
