HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
D. Transduction of hepatocytes. j 
1. Characteristics of the LNL6 vector and vector producing cell line . This work ’ 
will make use of the same vectors and vector producing cell line used previously for 
experiments in human subjects by Rosenberg et al (1990). The structures of these j_ 
materials are shown schematically in figure 3. The principles and methods for |P 
transducing hepatocytes are also similar to those established in other systems including ' 
human cells (Rosenberg, 1990) . || 
The LNL6 vector (Miller and Rosman, 1989) is a safety modified derivative of ant 
earlier vector N2 (Armentano et al, 1987) and is shown schematically in figure 3A. The |1 
N2 vector was constructed from the 5' and 3' terminal repeats of MMLV, an extended V f' 
sequence which included a portion of the 5' (amino terminal) reading frame for the i 
retroviral gag proteins and the neomycin resistance gene (NEO-R) from the bacteriophage i 
Tn5 (Southern and Berg, 1982). These segments were recombined in the plasmid pBR322 for 
cloning in E. coli . In order to improve the safety of the N2 vector, site-directed!* 
mutagenesis was performed to eliminate the initiation codon for the gag open reading j. 
frame and a splice site (Bender et al, 1987; Miller and Rosman, 1989) creating the LNL6 |' 
vector. This vector contains no retroviral reading frames and expresses the NEO-R gene i' 
from the promoter present in the 5' LTR. f; 
This clone was introduced into a packaging cell line PA317 to create the cell line 
PA317/LNL6 -c 8 . The PA317 cell line is a derivative of the NIH3T3 cell line which w^^s In 
transfected with a retroviral construct (figure 3B) which lacks the ij) sequence necessary; 
for retroviral packaging (Miller and Buttimore, 1986) and has other modifications 
designed to prevent recombination. Introduction of the LNL6 construct into the PA317 ’ 
cell line results in production of a vector which is composed of the capsule encoded by 
the pPAM3 construct, and contains the RNA transcript encoded by LNL6 (figure 1). Thej 
vector formed from this cell line can be harvested from the media of cells in culture. !; 
The PA317/LNL6-c8 subclone was selected as producing high titers of the defective!' 
retroviral vectors (2x10^ to 2x10*). Extensive studies on this cell line have'l 
demonstrated that the vector produced from the PA317/LNL6-c8 cell line is free of wildj' 
type (infectious) virus, other known murine viruses, or other known human pathogens! 
and passes a variety of safety tests required by the FDA (Appendix D) . j 
Frozen stocks of vector will be made available by Genetic Therapy Incorporated' 
(Bethesda, MD) for these experiments at no cost. The titering and certification of this 
material will be performed by GTI . j; 
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Recombinant DNA Research, Volume 14 
