5.22) 
a stop codon at the gag start codon. 
The LNL-6 producer cell line 
The LNL-6 producer cell line was created 
by transfection of the LNL-6 vector into 
the PA317 producer cell line (54) . The 
vector containing supernate obtained from 
the LNL-6 producer cell line is 
extensively tested for exogenous 
pathogens and replication-competent virus 
(see Section 7.3). 
The supernate (provided by Genetic 
Therapy, Inc.) to be used for human 
clinical use has passed all studies, and 
no pathogens were detected. The 
supernate is approved for clinical use by 
the U.S. Food and Drug Administration. 
5.3) Transduction Protocol 
At the time of marrow harvest the majority of the 
marrow will be untreated as not to affect or alter 
the therapeutic benefit and reconstitution 
potential of the transplanted marrow. At least 2xl0 8 
nucleated cells/kg must be obtained at harvest. The 
first 3 patients will have 10% of their marrow 
cells exposed to retroviral vector. If no untoward 
effects on marrow reconstitution is observed the 
portion of marrow treated will be escalated to 30% 
in the remaining patients. 
Untreated marrow will be frozen according to our 
current protocol (English et. al., Transfusion 
29:12-16, 1989) . 
The portion to undergo RMGT will first undergo 
Ficoll-Hypaque separation and the mononuclear cell 
fraction will be washed twice in phosphate buffered 
saline (PBS) . Cells will be exposed to the LNL-6 
vector at a ratio of 1 to 10:1 vector particles to 
cell. The mixture will be incubated at 37° for 4 
hours in the presence of protamine sulfate, a 
polycation which enhances transduction (55) . 
Marrow cells exposed to LNL-6 will be pelleted, 
washed twice in PBS, then frozen in a manner 
identical to the untreated marrow. Viable cell 
counts will be obtained prior to and after vector 
treatment. Preliminary data in the design of the 
transduction protocol can be found in Appendix 1. 
Recombinant DNA Research, Volume 15 
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