Experiments with mouse and human marrow with NEO positive 
marking vectors in the past also gave immediate and post-transplant 
frequencies of 5%. Our own experiments suggest that a similar 
frequency of marking occurs in the CML chronic phase marrow. The 
success of our marking experiment depends on the marked leukemic 
blast cells in the marrow competing effectively with unmarked 
leukemic and the non-leukemic populations present in the patient 
after transplant. Since most second chronic phase patients relapse, 
it is probable that the marked blast cells will increase in 
frequency and may become dominant in the patient following 
transplant if the autologous cells infusion is the major source of 
relapse. 
b4. TO WHAT EXTENT IS EXPRESSION FROM THE DESIRED GENE AND 
NOT THE SURROUNDING DNA? TO WHAT EXTENT DOES THE INSERTION MODIFY 
THE EXPRESSION OF OTHER GENES? 
These experiments are not designed to measure the expression 
of any parts of the marking vector transgenome. Although data to 
explicitly test the question of altered expression of genes 
surrounding the insertion site is not available, experience with 
transduction of NEO positive retroviral vectors as controls for 
retroviral-mediated insertions, which are designed to produce 
transformation via insertion of a transforming gene, have not led 
to an altered phenotype of the marked cell. A diploid complement 
of genes presumably protects the cell from the effects of loss of 
gene expression which may be increased by the vector integration. 
b5. IN WHAT PERCENT OF CELLS DOES EXPRESSION FROM THE ADDED 
DNA OCCUR? IS THE PRODUCT BIOLOGICALLY ACTIVE? WHAT PERCENT OF 
NORMAL ACTIVITY RESULTS FROM THE INSERTED GENE? 
On the basis of experiments reported in the literature, and 
our own data on CML presented in Table II and Figures 1-2, the 
frequency of cells in which a functional vector NEO transgenome is 
present is 10%. The NEO gene is not a gene found in mammalian 
cells and thus, all of the NEO is from the marking vector. 
b6. IS THE GENE EXPRESSED IN CELLS OTHER THAN THE TARGET 
CELLS? 
The NEO gene is not present in mammalian cells. 
2C. LABORATORY STUDIES PERTAINING TO THE SAFETY OF THE 
DELIVERY /EXPRESS ION SYSTEM : 
Two risks are recognized as potential complications from the 
marking of autologous bone marrow and peripheral blood cells with 
the LNL6 marking vector: 
Recombinant DNA Research, Volume 14 
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