sequence of that vector was submitted to the subcommittee 
previously. The LNL6 vector, a safety modified variant of N2 , was 
constructed by A. Dusty Miller (2) . It is replication incompetent 
and contains the NEO gene (2) . 
A procedure for incubation of the marrrow or peripheral blood 
which is similar to that utilized by Rosenberg and Anderson in the 
N2-TIL protocol will be used. The only difference is that human 
marrow and peripheral blood stem cells will be used instead of TIL 
cells. One third of 2 x 10^° nucleated cells (2 x 10®/kg) (separated 
from red cells and mature neutrophils by Ficoll gradients or 
continuous flow centrifugation) of CML patients will be exposed to 
the LNL6 vector by a protocol previously outlined by Malcolm 
Brenner (see below) : 
Transduction of Autologous Cells bv Malcolm Brenner (Protocol 
Approved bv the RAC on 2/4/91) 
This section is a slightly modified version of that written by 
Malcolm Brenner. 
1) 30% of the harvested autologous cells will be separated 
into a mononuclear cell preparation by Ficoll hypaque 
gradients or continuous flow centrifugation. The Ficoll 
hypaque marrow separation will reduce the total nucleated 
cells from 2 x 10’° to 4 x lo’. 
2) As shown in Table I, 30% of 4 x lo’ marrow cells (1.2 x 
lo’ marrow cells) will be used for transduction and 2.8 
X lo’ cells will not be exposed to the vector. Thirty 
percent of 1.4 x 10^*^ peripheral blood cells (4.2 x lo’ 
peripheral blood cells) will be used for transduction. 
The number of cells available for marking after Ficoll 
hypaque may be 1.2 x lo’ marrow cells and 4.2 x lo’ 
peripheral blood cells. 
3) Samples of the non-transduced and transduced cells will 
be cultured in methylcellulose (e.g. 10^ cells/cc) as 
outlined in step #9 below. 
4) The cells for exposure will be diluted to 1 x loV^l in 
Dulbecco Modified Eagles Medium. 
5) Genetic Therapy, Inc. clinical grade vector supernatant 
will be used for transduction which has been prepared, 
tested and stored by the procedures previously described. 
This supernatant contains between 5-20 x 10^ MOI 
(transducing) units/cc. 
6) After thawing, the vector supernatant will be added in a 
Category 2 laminar flow hood to autologous mononuclear 
cells (10 ml of supernatant to 10*^ cells) in Nunc flasks 
to reach a final concentration of 10 MOI/cell (10 ml/10^ 
cells) . 
Recombinant DNA Research, Volume 14 
[745] 
