Human Gene Therapy Subcommittee - 11/30/90 
said that he would present some updates on the data supplied with the initial 
protocol, and that Dr. Ihle would discuss details of the PCR reaction and inverted 
the PCR which will be used to determine signs of relapse and insertion sites in 
relapse. 
Dr. Brenner said that AML had been looked at in three different ways: 
1. Looking at clonigenic colonies by cytogenetics to determine 
morphological, histochemical, and genetic differences in the colonies 
which are transduced and grown in G418; 
2. PCR analysis of individual colonies to show the presence of the 790 base 
pair neomycin resistance gene to confirm the results of the experiments 
conducted by cytogenetics; and 
3. Use of a beta-galactosidase containing vector to identify transduced 
colonies. 
Dr. Brenner noted that in the nine leukemias studied, there is a transduction rate of 
approximately 10%. However, this transduction rate is variable and ranges from 0% 
to 25% transduction. 
Dr. Brenner presented data on a comparison of neuroblastoma cells transduced with 
the beta-galactosidase containing vector in which the transduction efficiency was 
analyzed by limiting dilution analysis. There was a range of transduction efficiencies 
ranging from 3 to 30%. These experiments indicated that if more than one cell in 
the marrow is responsible for causing relapse, it is possible that the protocol will be 
successful in identifying it. With 10% transduction efficiency, treating 30% of the 
leukemic cells would result in a 3% transduction of all leukemic cells. If 10 cells in 
a patient are responsible for the relapse, there is a 90% chance that the vector 
would be identified in 12 patients. 
Dr. Brenner cautioned that the finding of one relapse out of 12 patients would not 
be cause to alter the way in which clinical bone marrow transplantation is carried 
out, but that it would be cause to look at another 12 patients. If as many as 3 out of 
12 relapse, this result would be sufficient to suggest that purging should be 
performed in bone marrow transplants. 
Dr. Parkman asked if the possibility of non-cycling cells contributing to the relapse 
had been taken into account in Dr. Brenner's calculations. Dr. Brenner replied that 
the possibility had not been included in his analysis. Dr. Parkman asked whether 
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