since the present protocol deals with marking of CML cells 
which exhibit a higher percentage of cycling cells than the 
progenitor cells of the normal population, the frequency of 
integration may be higher than with normal progenitor cells. 
As shown in Table I, the number of residual blast crisis cells 
remaining in the 2 x 10^® autologous cells after re-induction of 
cytogenetic remission may be as high as 1% or as low as .001%. We 
estimate that if 1% of the cells are leukemic blasts at the time of 
marking, a total of 5.4 x 10* marked (marrow and peripheral blood) 
leukemic blast cells will be present in the autologous cells 
infused into the patient. 
b2 . What is the minimal level of gene transfer and/or 
expression which will be required for this gene transfer protocol 
to be successful in humans? How was this level determined? 
Previous data of Malcolm Brenner suggest that 10% of the 
leukemia cells in the marrow may be transduced by the LNL6 vector. 
These data had been reviewed in earlier applications (Brenner, 
2/91) . Thirty percent of the autologous cells are going to be 
exposed to the vector. Ten percent of the cells present in these 
autologous stem cell preparations will be marked by the LNL6 vector 
(see Table II and Figures 3-4) . As outlined in Tables I, IV, and 
V, the number of marked leukemic blast cells produced by this 
marking vector will be between 1.2 x 10* and 1.2 x 10^ in the marrow 
cells, and 4.2 x 10* and 4.2 x 10^ in the peripheral blood cells, 
depending on the percent of the marrow cells which are leukemic 
blasts is 1% or 0.001%. It is very unlikely that the conventional 
dose chemotherapy used to re- induce chronic phase could reduce the 
percentage of blasts below 0.001%. This produces a ratio of 
leukemic marked blasts which is between 1/3000 and 1/300,000 at the 
time of storage. This ratio will be higher at the time of relapse. 
Thus, the ratio of blasts is adequate to be detectable by PCR. 
In order to establish whether the NEO-positive cells appearing 
at relapse are leukemic or normal, PCR for bcr-abl mRNA will also 
be done on each colony. It is also possible that the frequency of 
transduction by the marking vector sequences may be much lower in 
some patients and variable from patient to patient. We will be in 
a position to detect this, through use of PCR for NEO and bcr-abl 
in a number of the CML patients. 
Since the PCR for bcr-abl mRNA assay will be done on all NEO 
positive colonies, we will be able to determine the relative 
frequency of the diploid and CML progenitors which are marked, and 
the number of different integration sites. This data could 
conceivably lead to important information concerning the clonal ity 
of human stem cell reconstitution in normal and leukemic 
progenitors as well. 
Recombinant DNA Research, Volume 14 
[757] 
