We are interested in developing therapies for the treatment of metabolic disorders. The 
model we have used is familial hypercholesterolemia (FH) which is caused by defects in the 
receptor that binds low density lipoproteins (LDL). 
B. Familial Hypercholesterolemia 
1. Genetics 
FH is an autosomal dominant disorder caused by abnormalities in the function or 
expression of LDL receptors (1,2). Patients who inherit one abnormal allele have moderate 
elevations in plasma LDL and suffer premature coronary heart disease (CAD). The prevalence 
of heterozygotes in most populations is 1 in 500 and they represent approximately 5% of all 
patients under 45 who have had a myocardial infarct. Patients with two abnormal LDL receptor 
alleles (homozygotes or compound heterozygotes) have severe hypercholesterolemia and life- 
threatening CAD. Features of FH that relate to the proposed ex vivo gene therapy will be 
summarized below. -In subsequent discussions, FH will refer to the more severe clinical 
syndrome that is associated with two abnormal LDL receptor alleles. 
The molecular basis of FH lies in the gene that encodes LDL receptors. Characterization 
of mutant alleles has revealed a variety of mutations including deletions, insertions, missense 
mutations, and nonsense mutations (2, 36). This genotypic heterogeneity leads to variable 
consequences in the biochemical function of the receptor which are classified in four general 
groups. Class I mutations are associated with no detectable protein and are often caused by gene 
deletions. Class 2 mutations lead to abnormalities in intracellular processing of the protein. 
Class 3 mutations specifically affect binding the ligand LDL, and class 4 mutations encode 
receptor proteins that do not cluster in coated pits. 
2. Metabolism 
An important issue in the development of gene therapy for FH relates to the specific 
somatic cell that is the target for gene transfer. We believe that the hepatocyte is the preferred 
target cell for gene therapy of FH. The rationale for this will be described in a broader 
discussion of the metabolic steps involved in the maintenance of cholesterol homeostasis in vivo. 
Virtually all somatic cells contain a group of highly regulated metabolic pathways 
capable of stabilizing intracellular cholesterol concentrations (2). Cholesterol can be produced 
by de novo biosynthesis or can enter the hepatocyte by receptor-mediated and non-receptor 
mediated uptake of lipoproteins and lysosomal hydrolysis of the component cholesterol esters. 
The enzyme that catalyzes the rate limiting step in de novo biosynthesis, HMG CoA reductase, and 
the receptor most responsible for lipoprotein catabolism, the LDL receptor, are both subject to 
feedback inhibition by intracellular cholesterol. Cholesterol has limited metabolic fates in most 
non-hepatic cells; it can be incorporated into membranes or stored intracellularly in the form 
of cholesteryl esters. The enzyme responsible for esterification of cholesterol, acylCoA: 
cholesterol acyltransferase, is activated in the presence of excess intracellular cholesterol 
(37). 
The liver modulates cholesterol homeostasis in vivo through a variety of metabolic 
functions that are uniquely expressed in its parenchymal cells. Hepatocytes secrete very low 
density lipoproteins (VLDL) which are the precursor to most other lipoproteins involved in 
endogenous metabolism of cholesterol (38). VLDL, a large triglyceride-rich lipoprotein, is 
converted to IDL through the action of lipoprotein lipase in capillary beds. IDL is recognized by 
the receptor for LDL through interactions with its two apoproteins, apo B100 and apo E. The 
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Recombinant DNA Research, Volume 15 
