1986 (8). There was a striking reduction in total serum cholesterol (76% decrease) after the 
transplant. The experience with liver transplantation vividly illustrates the importance of 
hepatic LDL receptor expression in modulating in vivo cholesterol metabolism. The spectacular 
metabolic improvement obtained following liver transplantation must be balanced with the 
associated perioperative mortality and substantial long term morbidity secondary to chronic 
immunosuppression. 
C. Preclinical Studies 
Our preclinical studies have focused in on two areas. The majority of our work has 
utilized a rabbit animal model for FH. We have used this model to develop the technology of 
liver-directed gene therapies and to assess efficacy of LDL receptor gene transfer in vivo. We 
have also used liver tissue from donors for orthotopic liver transplantation to develop methods 
for isolating and transducing human hepatocytes. 
1. Ex Vivo Gene Therapy in the Watanabe Heritable 
Hyperlipidemic Rabbit 
The animal used in our preclinical studies, called the Watanabe Heritable 
Hyperlipidemic rabbit (WHHL), was described in the 1970's by Dr. Watanabe (10). This 
animal demonstrates the clinical and metabolic abnormalities similar to those associated with 
FH in humans . The WHHL rabbit was instrumental in delineating key steps in the regulation of 
cholesterol metabolism and has been very useful in the design and testing of gene therapies for 
FH (11,13). 
The specific mutation responsible for the WHHL trait is an inframe deletion of 12 
nucleotides in the ligand binding domain of the receptor (12). This mutation leads to expression 
of a dysfunctional receptor protein that is inefficiently processed and unable to bind ligand (12, 
83). Quantitative LDL binding analyses of WHHL derived cells and tissues, and in vivo 
measurements of receptor dependent lipoprotein uptake, indicate that the receptor deficiency of 
this animal is essentially complete (<5% of control) (84-88). The metabolic consequences of 
this defect in rabbits closely resembles those described in patients with FH. In both cases, 
there is a selective accumulation of lipoproteins that contain apo B100, the polypeptide which 
is specifically recognized by the LDL receptor (i.e., VLDL, IDL and LDL). Lipoproteins not 
recognized by LDL receptor remain unchanged except for HDL which is slightly decreased in 
both WHHL rabbits and FH patients. The validity of the WHHL rabbit model was initially 
questioned when it was noted that the rabbits had elevated levels of both triglycerides and 
cholesterols, while FH patients had increased cholesterol but normal triglyceride. This was 
subsequently ascribed to unusually high triglyceride content in rabbit derived LDL (11). 
The full spectrum of clinical manifestations described in FH homozygotes are observed in 
the WHHL rabbit. Peripheral manifestations of severe hypercholesterolemia in the WHHL 
rabbit include lesions of the paws which resemble tendinous xanthomas (10). WHHL rabbits, 
like FH homozygotes, develop severe atherosclerosis attributable to severely elevated LDL that 
is restricted to the coronary arteries and proximal aorta (10). We have used the WHHL rabbit 
as a model to develop ex vivo approaches to liver directed gene therapy. 
The first step towards the development of these therapies was to design methods for 
isolating WHHL hepatocytes and efficiently transducing functional LDL receptor genes into the 
cells. A series of retroviral vectors that express the gene for human LDL receptor were 
constructed, each differing in the transcriptional elements used to drive LDL receptor 
expression (14). Helper-free amphotropic virus stocks representing each construct were then 
used to infect primary cultures of hepatocytes that were isolated from newborn WHHL rabbits. 
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