Recombinant DNA Advisory Committee - 09/14-15/92 
Dr. Parkman noted that the real question of interest is the transduction efficiency. 
Marker genes are readily detected in neoplastic cells by PCR when the starting 
population is 50%. What results are obtained when a patient is in remission where the 
frequency of neoplastic cells could be 2-3 logs lower? Will the neo R be detectable? Dr. 
Deisseroth explained that the goal of the protocol is to assess the presence or absence of 
the markers at the time of relapse. At relapse, the ratio of normal to leukemic cells will 
be 1:1. Dr. Parkman stated that since most CLL cells are not cycled, the transduction 
efficiency could be 1 in 1,000 cells. The probability of transduction could be 1-2 logs less 
than predicted. Dr. Deisseroth said that CLL is a disease in which cells accumulate 
rather than result from a highly proliferative population. However, there is a low 
frequency of CLL cells which are proliferating. At the time that the CLL population will 
be transduced with the vector, the culture conditions will promote proliferation in this 
small fraction of cells. It is this population of proliferating cells which contribute to 
relapse. 
Dr. Parkman inquired about the incubation conditions. Dr. Deisseroth explained that 
the cells will be transduced in Dexter culture without hydrocortisone on stromal 
monolayers. Basically, the Whitlock-Witte culture system. The ABM cells are incubated 
on stromal monolayers that have been irradiated with and without hydrocortisone. 
Hydrocortisone promotes the growth of myeloid cells while the absence of 
hydrocortisone promotes lymphoid cell growth. The retroviral vector will be added to 
the cells at a ratio of 10 infectious particles per cell for 72 hours. 
Dr. Deisseroth explained that both FACS analysis and fluorescent in situ hybridization 
(FISH) will be used to distinguish abnormal cells from normal cells. FISH analysis 
requires the use of a DNA probe that is specific for chromosome 12. The presence of 
differentiation antigens allows for the selection of abnormal cells by FACS and analysis 
of the marker gene by FISH and PCR. 
Dr. Deisseroth explained the cell fractionation process. The positive selection of normal 
cells is accomplished by incubation with the CD34 monoclonal antibody conjugated to 
biotin and passage through an avidin column. The biotin binds to avidin and the 
CD34( + ) cells remain in the column. This process enriches for early progenitor cells by 
two logs, and there is a three log reduction in neoplastic cells because neoplastic cells do 
not possess the CD34 antigen. The negative selection procedure eliminates CD19(-) 
leukemic cells. Magnetic beads conjugated to CD19(-) are incubated with the enriched 
CD34( + ) cell population at 4°C resulting in a two log reduction in the abnormal cell 
population. Therefore, the overall enrichment frequency of the normal cells is two logs, 
and the depletion of abnormal cells is five logs. 
Dr. D. Miller asked if CD 19 binds CLL progenitor cells. Dr. Deisseroth explained that 
the progenitor for CLL has never been isolated. Dr. D. Miller asked if a colony forming 
[262] 
Recombinant DNA Research, Volume 16 
