The transduced hepatocytes produced dramati- 
cally higher levels of mouse PAH mRNA than did 
mock-infected hepatocytes. The PAH mRNA was 
translated efficiently into PAH protein that is capa- 
ble of converting phenylalanine to tyrosine in vitro. 
These results demonstrate that the PAH-deficient 
mouse hepatocytes can be readily reconstituted by 
retroviral-mediated gene transduction. (This por- 
^ tion of the work was supported in part by a grant 
from the National Institute of Diabetes and Digestive 
and Kidney Diseases, National Institutes of Health.) 
The next step is to develop technologies for the 
efficient transfer of therapeutic genes into living an- 
imals in order to correct a deficient phenotype. The 
current strategy for hepatic gene therapy involves 
the isolation of primary hepatocytes from a resected 
liver lobe and transduction of therapeutic genes 
into the cultured cells, followed by autologous he- 
patocellular transplantation. The ex vivo approach 
has been reported in mice, rabbits, and dogs, but is a 
complex procedure in its entirety. Thus a simple 
method for direct gene delivery into hepatocytes in 
vivo was developed. The procedure involves partial 
hepatectomy followed by portal vein infusion of re- 
combinant retroviral vectors. 
Histologic analysis of hepatocytes after in vivo 
delivery of a recombinant retrovirus bearing the 
Escherichia coli jS-galactosidase gene showed that 
1 -2% of the parenchymal cells were transduced. Di- 
rect hepatic transfer of human aj-antitrypsin cDNA 
under the transcriptional direction of the albumin 
promoter-enhancer led to constitutive expression of 
the human protein in the sera of mouse recipients at 
concentrations of 30-1,400 ng/ml for at least 6 
months. The experimental animals showed no signs 
of illness, and histologic analysis of the liver re- 
vealed no evidence of pathologic abnormalities. 
The persistent expression of a foreign gene in the 
recipient liver suggests that the in vivo approach is 
an attractive alternative to hepatic gene therapy. 
(This portion of the work was supported in part by a 
grant from the National Heart, Lung, and Blood Insti- 
tute, National Institutes of Health.) 
Dr. Woo is also Professor of Cell Biology, Molec- 
ular Genetics, and Pediatrics at Baylor College of 
Medicine. 
Articles 
Eisensmith, R.C., and Woo, S.L.C. 1991. Phenylke- 
tonuria and the phenylalanine hydroxylase gene. 
Mol Biol Med 8:5-i8. 
Eisensmith, R.C., and Woo, S.L.C. 1992. Molecular 
basis of phenylketonuria and related hyperphe- 
nylalaninemias: mutations and polymorphisms in 
the human phenylalanine hydroxylase gene. Hum 
Mutat 1:13-23. 
Goltsov, A.A., Eisensmith, R.C., and Woo, S.L.C. 
1992. Detection of the XmnI RFLP at the human 
PAH locus by PGR. Nucleic Acids Res 20:927. 
Kay, M.A., Baley, P., Rothenberg, S., Leland, P., 
Fleming, L., Ponder, K.P., Liu, T.J., Finegold, M., 
Darlington, G., Pokorny, W., and Woo, S.L.C. 
1992. Expression of human a j -antitrypsin in dogs 
after autologous transplantation of retroviral 
transduced hepatocytes. Proc Natl Acad Sci USA 
89:89-93. 
Kay, M.A., Ponder, K.P., and Woo, S.L.C. 1991. Hu- 
man gene therapy: present and future. Breast 
Cancer Res Treat 21:83-93- 
Ledley, F.D., Woo, S.L., Ferry, G.D., Whisennand, 
H.H., Brandt, M.L., Darlington, G.J., Demmler, 
G.J., Finegold, M.J., Pokorny, W.J., Rosenblatt, H., 
Schwartz, P., Moen, R.C., and Anderson, W.F. 
1991- Hepatocellular transplantation in acute he- 
patic failure and targeting genetic markers to he- 
patic cells. Hum Gene Ther 2:331-358. 
Li, J., Eisensmith, R.C., Wang, T., Lo, W.H.Y., 
Huang, S.Z., Zeng, Y.T., Liu, S.R., and Woo, S.L.C. 
1992. Identification of three novel missense PKU 
mutations among Chinese. Genomics 13:894- 
895. 
Liu, T.J., Kay, M.A., Darlington, G.J., and Woo, 
S.L.C. 1992. Reconstitution of enzymatic activity 
in hepatocytes of phenylalanine hydroxylase- 
deficient mice. Somat Cell Mol Genet 18:89-96. 
Reichardt, J.K.V., Belmont, J.W., Levy, H.L., and 
Woo, S.L.C. 1992. Characterization of two mis- 
sense mutations in human galactose- 1 -phosphate 
uridyltransferase: different molecular mecha- 
nisms for galactosemia. Genomics 12:596-600. 
Reichardt, J.K. v.. Levy, H.L., and Woo, S.L.C. 1992. 
Molecular characterization of two galactosemia 
mutations and one polymorphism: implications 
for structure-function analysis of human galac- 
tose-! -phosphate uridyltransferase. Biochemistry 
31:5430-5433. 
Reichardt, J.K.V., Packman, S., and Woo, S.L.C. 
1991 . Molecular characterization of two galacto- 
semia mutations: correlation of mutations with 
highly conserved domains in galactose- 1- 
phosphate uridyl transferase. Am f Hum Genet 
49:860-867. 
Svensson, E., Eisensmith, R.C., Dworniczak, B., von 
Dobeln, U., Hagenfeldt, L., Horst, J., and Woo, 
S.L.C. 1992. Missense mutations causing mild hy- 
perphenylalaninemia associated with DNA haplo- 
type. Hum Mutat 1:129-137. 
Wang, T., Okano, Y., Eisensmith, R.C., Lo, W H Y., 
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