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PATHOGENESIS AND TREATMENT OF INHERITED DISEASES 
James M. Wilson, M.D., Ph.D., Assistant Investigator 
The overall goal of Dr. Wilson's research is to de- 
fine the pathogenesis of human inherited diseases 
and to develop therapies based on somatic gene 
transfer. In the past year he has made progress in two 
model systems. 
Familial Hypercholesterolemia 
An important area of research in Dr. Wilson's labo- 
ratory has been the development of gene replace- 
ment therapies based on gene transfer into the liver. 
An inherited deficiency of the receptor for low- 
density lipoproteins (LDLs), which leads to the fa- 
milial hypercholesterolemia (FH) syndrome, has 
been used as a model for such development. Critical 
to this effort has been the availability of an animal 
model for FH, the Watanabe heritable hyperlipid- 
emic (WHHL) rabbit. The initial strategy for genetic 
treatment of FH is based on transplantation of autolo- 
gous hepatocytes that have been genetically 
corrected ex vivo with recombinant retroviruses. 
The feasibility of ex vivo gene therapy of FH was 
established in experiments with WHHL rabbits. He- 
patocytes harvested from these animals were plated 
in primary cultures and exposed to recombinant ret- 
roviruses capable of transferring a functional rabbit 
LDL receptor gene efficiently. These genetically 
modified cells were then injected into the portal 
vein of the animal of origin. The transplant recipi- 
ents demonstrated substantial levels of recombinant 
LDL receptor RNA in their liver for the duration of 
the experiment, which was terminated six months 
after initial cell transplantation. Transplant recipi- 
ents realized a 20-50% diminution in serum choles- 
terol, which persisted for at least four months. 
The results obtained with the WHHL model en- 
couraged Dr. Wilson and his co-workers to under- 
take a series of experiments in preparation for a 
clinical trial of gene therapy against homozygous 
FH. Techniques for isolating human hepatocytes 
were established, and recombinant retroviruses ca- 
pable of transducing a fully functional LDL receptor 
gene were produced. Pilot experiments were per- 
formed in baboons to assess the safety and toxicity of 
ex vivo, liver-directed gene therapy. A clinical pro- 
tocol for treating patients with severe homozygous 
FH, based on transplantation of autologous, geneti- 
cally corrected hepatocytes proposed by Dr. Wilson 
and his colleagues was approved by the Recombi- 
nant DNA Advisory Committee of the National Insti- 
tutes of Health and the Food and Drug Administra- 
tion. The first patient was treated in June 1992. 
Grants from the National Institutes of Health pro- 
vided support for the program in FH gene therapy. 
An alternative approach to liver-directed gene 
therapy of FH that may be more effective and less 
morbid is the direct delivery of a functional LDL 
receptor gene to hepatocytes in vivo. One promis- 
ing approach, which Dr. Wilson and his colleagues 
are pursuing, is the development of gene transfer 
substrates composed of DNA-protein complexes. A 
ligand for the liver-specific asialoglycoprotein re- 
ceptor is conjugated to DNA of an expression vector. 
Initial experiments analyzed the expression of a re- 
porter gene in tissues of rats that had been injected 
intravenously with a DNA-protein complex. 
Strategies for obtaining stable expression of the 
transgene in vivo have been identified. This is ac- 
complished by subjecting the animal to partial hepa- 
tectomy immediately after injection of the complex. 
Analysis of DNA from liver tissue of animals that 
stably express the recombinant gene revealed some 
unexpected results. The recombinant gene abun- 
dantly persists in liver tissue for at least four months 
as episomal DNA that retains the structural and meth- 
ylation pattern of the input plasmid. 
Studies are under way to use this approach to 
correct the genetic defect in several animal models 
of human diseases, including LDL receptor gene 
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