The short term fate of the transplanted cells was studied by in situ hybridization using a 
cRNA probe specific for vector-derived sequences in the recombinant transcript. Analysis of 
liver sections revealed transduced hepatocytes in a periportal distribution. This is consistent 
with the hepatocytes seeding in sinusoids soon after leaving the portal venule. 
These studies illustrate several important points regarding the feasibility of ex vivo 
gene therapy for FH. Reconstitution of only 4% of LDL receptor expression at the RNA level 
leads to a 30-40% decline in total serum cholesterol. This observation is consistent with 
studies in FH homozygotes which have demonstrated an inverse correlation between residual 
LDL receptor activity and serum cholesterol (41). 
We next sought to modify the protocol to achieve more prolonged, if not permanent, 
metabolic improvement. Our strategies to improve the therapy are based on the hypothesis that 
the rapid deterioration of LDL receptor function in the allogeneic ex vivo experiments is due to 
rejection caused by immune responses to the allogeneic cells or to the human LDL receptor 
protein. 
We have overcome these potential problems in a second series of experiments (18). A 
full-length cDNA clone for rabbit LDL receptor was isolated and cloned into retroviral vectors. 
High titer amphotropic producers were made with a vector that expresses rabbit LDL receptor 
from a (3-action promoter. Viruses that express the lacZ gene from the viral LTR were used in 
control experiments. A group of WHHL rabbits underwent partial hepatectomies and the liver 
tissues were used to prepare hepatocytes and establish primary cultures. The cultured 
hepatocytes were exposed to the second generation viruses described above, harvested, and 
transplanted into the spleen of the animals from they were derived. The majority of the cells 
immediately passed through the spleen into the portal circulation and seeded the liver. Animals 
transplanted with LDL receptor transduced hepatocytes (N=5) demonstrated a 30-50% 
decrease in total serum cholesterol that persisted for the duration of the experiment (122 
days). No significant change in serum cholesterol was noted in animals that received lacZ 
transduced hepatocytes (N=7). RNase protection assays demonstrated recombinant derived LDL 
receptor RNA in liver and spleen tissues harvested up to 6.5 months after transplantation. No 
diminution in recombinant RNA was noted during this time period. Albumin expressing cells 
were detected in spleens of transplant recipients indicating that the transplanted cells retained 
differentiated function in vivo. Transplant recipients did not develop a serologic response to 
wild type rabbit LDL receptor protein as determined by Western blot analysis. 
These studies represent a substantial improvement over previous experiments of 
allogeneic hepatocyte transplantation [i.e., WHHL hepatocytes expressing human LDL receptor 
protein (16) or hepatocytes derived from a wild type rabbit(17, 89)] in WHHL rabbits which 
demonstrated transient metabolic effects in the absence of immunosuppressive therapy . The 
only available prolonged treatment of FH, allogeneic orthotopic liver transplantation, also 
requires immunosuppression with its associated morbidity (5-8). The major advantage of ex 
vivo gene therapy with autologous hepatocytes, as demonstrated in this animal model, is that 
long term function can be achieved in the absence of immunosuppressive therapy. Our 
experiments also address the potential complication of destructive immunological responses to 
the recombinant LDL receptor protein in recipients that had not previously been exposed to LDL 
receptor proteins. Functional stability of the transduced cells in vivo and the absence of a 
serological response to the wild type receptor suggests that immunological rejection may not be 
a confounding or limiting problem. 
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Recombinant DNA Research, Volume 15 
