HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
2. Transduction of hepatocvtes FDA certified, frozen supernatant containing the : 'i 
LNL6 vector will be provided by GTI free of charge. This material will be stored at - . 
70°C and thawed prior to use. This material will be filtered through Corning 0.22 
micron filters and primary human hepatocytes will be transduced by replacing the 
hepatocyte culture media with a 1:1 mixture of the media containing the vector and fresh i 
hepatocyte culture media with protamine added to a final concentration of 5 ug/ml 
(Cornetta and Anderson, 1989) . Cells will be incubated for four to six hours at 37° * 
under 5% CO^, washed with PBS, and then fed with fresh hepatocyte culture media. This 
procedure will be repeated at 12 hour intervals for 48 hours. Cultures will then be 
allowed to continue for an additional 24 hours before cells are harvested for 
transplantation. As a control, each batch of vector used will be plated on NIH3T3 cells ^ 
to confirm the apparent titer. 1 
For transplant, the transduced cells will be washed extensively in phosphate Aj 
buffered saline (PBS), trypsinized with parvovirus free trypsin from the culture plates, J 
and resuspended in PBS. The wash will involve at least 6 washes of 10 mis each. If ' 
there is a residual of lOOul vector containing media on each plate, this wash will ' 
result in a dilution of >10® and should be free of the vector which has an initial titer 
of 10*. Following trypsinization, cells will be allowed to settle and will be 
resuspended in isotonic saline solution containing 5% human albumin for infusion. 
I 
The efficiency of transduction will be monitored by selection of control plates of 
hepatocytes in the presence of G418 (800 ug/ml) and Southern blot on selected and un- ‘'i; 
selected cells. The efficiency of transduction will be quantitated by scanning 
densitometry of Southern blots against control cells containing dilutions of cell lines T 
containing the LNL6 provirus with one provirus/diploid genome. (It should be noted that S 
the efficiency of transduction in each individual batch of hepatocytes will not be known B| 
prior to implantation.) |l| 
Each wash solution will be tested for the presence of residual vector by adding W 
aliquots to NIH3T3 cells in the presence of 8ug/ml polybrene and selecting for neomycin {■ 
(G418) resistance. Aliquots of transduced cells, the media containing the vector, and 1 
select wash solutions will be tested for the presence of wild type virus by marker * 
rescue. These experiments will be performed by adding aliquots of solutions and 
polybrene to cells containing a provirus with a B-galactosidase marker gene. This 
supernatant will then be used to infect NIH3T3 cells. Aliquots of media will be tested 
for sterility in standard bacterial assays as well as for endotoxin. (It should be 
noted that the results of these tests (especially titer) may not be available at the 
time the transplant is performed.) 
3. Virology methods . We will assay the apparent titer of the LNL6 vector in 
NIH3T3 or rat 208F cells or primary murine hepatocytes using methods described 
previously (Miller et al, 1986; Ledley et al, 1986; 1987a). This involves transducing 
NIH3T3 or rat 208F cells with vector in the presence of 8 ug/ml polybrene, selection of 
colonies expressing the NEO-R gene with G418 (1 mg/ml). We can also assay for 
retroviral RNA in tissue culture media, wash solutions, and patient samples using 
coupled reverse transcriptase and PGR to amplify recombinant sequences as described 
[812] 
Recombinant DNA Research, Volume 14 
