4. Level and Expression of Transferred Gene 
The clinical correlations described in the previous section are of potential significance 
for gene therapy of FH. The ultimate goal of gene therapy in this disease is to replace the normal 
complement of LDL receptor activity in all hepatocytes thereby normalizing total serum 
cholesterol. This, however, will be a difficult task because of the large number of potential 
target cells in a human liver. The relevant question in terms of this protocol is whether partial 
reconstitution of LDL receptor function may be therapeutic. This issue will be further 
discussed in section IV. 
Another issue in the development of gene therapy for FH is the importance of regulating 
the expression of the transgene. The normal endogenous LDL receptor plays an important role 
in the maintenance of cellular cholesterol balance. Excess accumulation of intracellular sterol 
leads to suppression of exogenous sterol uptake by direct repression of LDL receptor gene 
transcription (51, 52). Most LDL receptor gene transfer systems under consideration for gene 
therapy utilize transcriptional signals that would result in constitutive expression of the LDL 
receptor gene (14, 53). A potential problem with this approach is that the constant 
unregulated uptake of LDL could lead to excessive accumulation of intracellular cholesterol and 
cholesteryl esters which may be toxic to hepatocytes. 
Previous LDL receptor gene transfer experiments have used constitutive or inducible 
promoters to drive the expression of the transferred gene. LDL receptor genes have been 
introduced into a variety of cell lines by transfection (54, 55) or retroviral infection (14, 
53). Constitutive and high level expression of the LDL receptor transgene in these cell lines 
had no reported effect on cell growth or function. However, transfected cell lines may not be the 
best experimental system to test the effects of excessive accumulation of intracellular 
cholesterol because they have high requirements for exogenous cholesterol by virtue of their 
ongoing cell division. More recently, we have used recombinant retroviruses to transfer a 
functional human LDL receptor gene into hepatocytes from a rabbit that is genetically deficient 
in LDL receptor expression (see Section II.C.1, Ref. 14). Constitutive overexpression of LDL 
receptor (greater than 4-fold over normal levels) in these infected cells had no detectable 
effect on cell viability or morphology during the time course of these experiments (48 to 72 
hours following the retroviral infection). More importantly, the cells persisted and continued 
to express high levels of the recombinant transcript after transplantation into autologous LDL 
receptor deficient rabbits (see below). 
Another informative gene transfer experiment was recently described by Hofmann et al., 
where a human LDL receptor gene, driven by the metallothionine promoter, was introduced into 
the germ line of a mouse (56, 57). Several transgenic animals that resulted from this 
experiment were shown to express high levels of human LDL receptor in liver when the 
transgene was induced with CdS04. Under these conditions, the transgenic animals demonstrated 
markedly increased catabolism of 12 ^l-labeled LDL and a virtual disappearance of serum LDL; 
no ontoward effects of this hepatic overexpression of LDL receptor were described (56). In 
addition, hepatic overexpression of the LDL receptor transgene prevented the development of 
hypercholesterolemia in response to a high lipid diet (57). 
5. Alternative Therapies 
A variety of surgical and pharmacologic therapies have been tried in the treatment of 
homozygous FH. These include diet, pharmacologic agents (58-63), portacaval shunts (3, 64, 
65), plasma exchange (4, 66-74), LDL apheresis (75-77), and orthotopic liver 
transplantation (5-9). The response of FH homozygotes to drugs is dependent, in part, on the 
residual function of LDL receptor. Intensive pharmocologic and dietary therapy has been 
[ 158 ] 
Recombinant DNA Research, Volume 15 
