Molecular Genetics and Studies Toward Gene Therapy 
for Metabolic Disorders 
lived for only a few weeks in the recipient ani- 
mals. When the hepatocytes were returned to the 
liver by direct injection into the portal vein or the 
spleen of recipient mice, however, the cells mi- 
grated to the liver and incorporated themselves 
into the parenchyma. They not only lived for as 
long as the mice did but also continued to func- 
tion as liver cells in the transplanted animals. Us- 
ing liver cells isolated from a mouse strain that is 
deficient in hepatic PAH activity, we demon- 
strated that enzymatic activity was reconstituted 
in these cells after retroviral-mediated gene 
transfer. The PKU mouse model will be critically 
important to test the efficacy of our hepatic gene 
transfer and hepatocyte transplantation protocols 
for the correction of PKU in the future. 
To develop a larger animal model for somatic 
gene therapy, with technologies that may be di- 
rectly applied to human patients in the future, we 
selected a colony of hemophilic dogs. Methods 
were developed to obtain a liver lobe from nor- 
mal dogs by partial hepatectomy and to disperse 
the hepatocytes into single cells in culture. In- 
stead of a few million cells (which we obtained 
from the mouse), several billion cells were ob- 
tained from a single canine liver lobe. The iso- 
lated hepatocytes were transplanted back into the 
same animal by direct injection into the spleen. 
Alternatively, a catheter was inserted into the 
splenic vein with a subcutaneous port to permit 
direct external injection of hepatocytes into the 
port. It was observed that a billion hepatocytes 
can be easily transplanted, and greater than half 
of these cells migrate to the liver and survive in 
the animal for a minimum of four months. Using a 
recombinant retroviral vector containing the hu- 
man coagulation factor IX gene to transduce nor- 
mal canine hepatocytes, we also observed high 
levels of human factor IX protein in the culture 
media. With these technological developments, 
we shall attempt to correct the genetic deficiency 
in hemophilia B dogs by somatic gene therapy. 
Prenatal diagnosis of PKU and disease progno- 
sis by gene analysis were supported by a grant 
from the National Institutes of Health. Somatic 
gene therapy research on the PKU mouse and he- 
mophilia B dog models is also supported in part 
by a grant from the National Institutes of Health. 
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