The vector system that will be used to transfer the neo gene in this 
pilot study is essentially the same as that used at NCI in the N2-TIL 
human gene transfer trial (37). This system consists of the GINa 
vector packages in an amphotrophic producer cell line (PA317). A 
similar vector, LNL6, is currently being used by Dr. Malcolm Brenner 
at St. Jude's Research Hospital to transduce bone marrow stem cells 
from autografts in children with AML and neuroblastoma. 
Preliminary results from this ongoing pilot study indicate that the 
transduced neo gene has been demonstrated in the bone marrow and 
peripheral blood at 4 weeks in one patient with neuroblastoma post- 
ABMT (reported at the NIH Gene Therapy meeting, December 4, 1991). 
The LNL6 and GINa supernatants are helper virus-free and no 
secondary malignancies have been reported in the human trials to 
date. 
The GINa supernatant has been approved by the FDA for experimental 
infection of human cells. GINa will be used in several clinical 
studies in the near future. GIN (also called GINa) is a retroviral 
vector-containing supernatant used for transferring the bacterial 
gene neophospho-transferase (NEO R ) into cells. This gene product 
confers resistance to the neomycin analogue, G418. The GIN 
producer cell line contains the vector plasmid (pGIN) integrated into 
PA317 (packaging) cells. The plasmid, PGIN, was constructed at 
Genetic Therapy, Inc. as a complement to LNL6 and provides some 
additional safety factors and cloning advantages over the LNL6 
plasmid made in the laboratory of A. Dusty Miller (BB-MF 3886). The 
deletion of unnecessary 3' sequences just 5' to the 3' LTR was 
accomplished in the pGI plasmid to eliminate any homology in this 
region with sequences that are also found in the PA317 cells from 
the pPAM3 plasmid containing the structural genes for the retroviral 
vector. The pGI plasmid contains a multicloning sequence which 
provides a convenient cloning site into which multiple genes or 
sequences can be inserted in a reproducible and consistent manner. 
To construct pGIN, a truncated (minimal 3' untranslated sequences) 
neo R gene is inserted into the multicloning site. Specifically, a 852 
base sequence containing the coding sequences for the neo R gene 
from the EcoRI site to the Asu II site was made by blunt ended 
ligation with DNA polymerase into pGI that had been cut with SnaBI. 
Recombinant DNA Research, Volume 16 
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