b3 . Explain in detail all results from animal and cultured 
cell model experiments which assess the effectiveness of the 
delivery system (see part 2a above) in achieving the minimally 
required level of gene transfer and expression (2b above) . 
As described above, morphology, fluorescence in situ 
hybridization (FISH) , and PCR will be used to measure the level of 
leukemia cells in the autologous cells used for autologous 
transplantation. Marrow morphology and Southern blotting, as a 
criterion of CML blast cell detection, is only good to a level of 
leukemia cells which is greater than 1 leukemia cell in 100 normal 
cells (1%) . The use of the FISH technique, which is based on the 
enumeration of chromosome 22 specific fragments of DNA (3 in the 
CML cells which harbor a balanced translocation between chromosomes 
9 and 22, and 2 in the normal hematopoietic cell) in interphase 
cells is being developed in our laboratory to permit definitive 
evaluation of the ratio of leukemia to normal cells down to a ratio 
of 1 leukemic cell/100,000 normal cells. PCR analysis can detect 
even lower levels of leukemia cell contamination. The range of 
contamination of the marrow may range anywhere between a maximum of 
1/100 (leukemic to normal) when defined as morphologically 
identifiable blasts, to a level of 1/10,000. Assuming that 4 x id’ 
marrow cells and 1.4 x 10^° peripheral blood (as shown in Table I, 
this is the number left after 2 x 10^° marrow and 6 x 10^° peripheral 
blood cells are subjected to Ficoll hypaque or continuous flow 
centrifugation) cells are transplanted, that 30% of these cells 
will be exposed to the vector and that 10% of these cells will be 
marked by the vector, the absolute number of leukemic blast cells 
marked in the autologous marrow infused will be between 5.4 x 10^ 
(if the leukemic blasts are 1% of the total) and 5.4 x 10^ (if only 
0.001% of the total cells infused are blasts). 
Thus, the ratio of the number of marked leukemic blast cells 
to other cells could be as high as 1/3000 and as low as 1/300,000. 
In the worst case, the presence of virally-marked blastic leukemia 
cells is on the borderline of detectability by PCR at the time of 
transplant. However, if relapse occurs, the ratio will be higher 
and clearly detectable by PCR. 
From these calculations, which are based on our observed 
marking frequency of chronic phase marrow with LNL6, the proposed 
marking studies would generate sufficient quantities of marked 
leukemic blasts to answer whether relapse is occurring from 
leukemia cells present in the infused autologous cells or remaining 
in the systemic circulation after TBI, VP-16, and cytoxan. On the 
basis of the number of unique integration sites, one can determine 
if relapse is a clonal phenomenon or not and whether it comes from 
the infused cells or residual systemic disease. Data from human 
leukemia cell lines vary from 1 to 50% in marking frequency with 
LNL6. Malcolm Brenner found a 10% frequency of marking in AML 
marrow in data supplied to the committee in November 1990, which 
agrees with our data on CML. 
[758] 
Recombinant DNA Research, Volume 14 
