The efficiency of transduction, as measured by Southern analysis of integrated proviral 
sequences, ranged from 20% to 100%. Expression of human LDL receptor was analyzed by blot 
hybridization analysis of total cellular RNA and by biochemical and in situ analyses of 
transduced cultures for receptor function. The vector in which the expression of LDL receptor 
was driven by the viral long terminal repeat sequence produced the greatest quantity of LDL 
receptor RNA and protein in WHHL hepatocytes; LDL receptor activity approached normal 
levels in these cultures. 
The next and more difficult step was to develop methods for transplanting LDL receptor- 
expressing hepatocytes into WHHL rabbits in a way that the cells function and persist. 
Hepatocytes were isolated from an outbred strain of rabbits that express normal levels of LDL 
receptor (New Zealand white strain) and transplanted into WHHL recipients by direct injection 
into the portal vein or into the peritoneal cavity attached to microcarrier beads (17). 
Transplantation of NZW hepatocytes using either method led to a 25% decrease in total plasma 
cholesterol over a 3 to 4 day period with a gradual return to pretreatment levels after 10 days. 
The resulting decrease in total plasma cholesterol was due to coordinate decreases in 
lipoproteins that are known ligands for the LDL receptor. Transplantation of allogeneic WHHL 
hepatocytes into WHHL recipients did not lead to a decrease in total plasma cholesterol. 
These experiments indicate that ectopically placed hepatocytes (in liver sinusoids or in 
the peritoneal cavity) can function with respect to lowering cholesterol, but only in a transient 
manner. Wiederkehr et. al. have demonstrated more prolonged function of transplanted NZW 
hepatocytes in WHHL rabbits when the animals were chronically immunosuppressed with 
cyclosporin (89). This suggests that the deterioration in LDL receptor function which occurred 
in our study was due to rejection of the cells. This could be due to major histocompatability 
complex (MHC) incompatabilities between the two strains of rabbits or an immunological 
response to the recombinant derived LDL receptor protein on the donor-derived cells. 
The feasibility of ex vivo gene therapy in the WHHL rabbit was demonstrated in a 
modification of the allogeneic experiments described above (16). Liver was harvested from a 
WHHL rabbit and hepatocytes were harvested and plated in primary culture. Recombinant genes 
were transduced into 10-20 % of the cultured hepatocytes which were harvested 4 days after 
the initial plating and transplanted into a group of WHHL recipients. Our colony of WHHL 
rabbits are outbred so this would constitute an allogeneic transplant.. There were two 
experimental groups: animals that received allogeneic WHHL hepatocytes that were either 
mock-infected (N=6) or infected with recombinant retroviruses that contain a functional 
human LDL receptor gene (N=7). 
Analysis of the primary cultures prior to transplantation indicated that the bulk 
population of cells expressed levels of recombinant LDL receptor protein and RNA in excess of 
that found in NZW hepatocytes. The metabolic consequences of hepatocyte transplantation was 
assessed by measuring changes in total serum cholesterol. No significant change in serum 
cholesterol was noted in animals that received mock infected hepatocytes. However, a 
substantial (30-40%) but, again, transient decline in serum cholesterol was achieved after 
transplantation of the LDL receptor transduced cells. 
Transplant recipients were further characterized with respect to recombinant gene 
transfer and expression. Liver tissue was harvested 10 minutes, 24 hours, and 19 days after 
transplantation of the LDL receptor transduced hepatocytes and analyzed for the presence of 
recombinant-derived RNA by RNase protection and proviral DNA by the polymerase chain 
reaction. Recombinant human LDL receptor RNA was detected 10 minutes and 24 hours after 
transplantation at 2-4 % the level of the endogenous transcript. Proviral DNA was also detected 
at both time points. Analysis of tissue harvested 19 days after transplantation detected neither 
the proviral DNA or the recombinant RNA. 
Recombinant DNA Research, Volume 15 
[161] 
