HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
concentrations in the liver than in other tissues. While there is evidence that 
substrates may be transferred to adjacent cells in culture and metabolized, there is no 
evidence that such metabolite transfer occurs across a fluid phase. Thus constitution 
of enzyme at remote sights might not mitigate the hepatotoxicity which is characteristic 
of the fulminant life-threatening disease (Ledley et al , unpublished data). 
There may be technical advantages to hepatic gene therapy, since this approach will 
involve introducing genes into easily purified, fully differentiated cell with 
considerable proliferative potential. It is possible that patients with severe liver 
damage, such as those selected for this study, will have high endogenous levels of 
factors which stimulated hepatic regeneration, and that the engrafted cells will exhibit 
substantial proliferation after transplantation. Moreover, gene expression in these 
cells may not be confounded by problems of developmental regulation and variable stem 
cell activation which are observed in bone marrow experiments. As a result, several 
laboratories are actively engaged in developing methods for hepatic gene therapy (Ledley 
et al, 1987a; Peng et al, 1988; Wilson et al , 1988a; 1988b; 1990; Wolff et al, 1987a; 
1987b; Anderson et al, 1989). 
This discussion of hepatic gene therapy is intended to illustrate why we believe 
that clinical trials assessing the applicability of gene transfer techniques for 
transducing hepatic cells prior to HGT would yield knowledge of value. 
H. Transduction of hepatocytes with recombinant retroviral vectors and expression of 
recombinant genes . 
Early studies by Jaenisch and his colleagues suggested that the liver is relatively 
resistant to infection by wild type retroviruses in vivo after 8 days gestation 
(Jaenisch, 1980; Stuhlmann et al, 1984), even if partial hepatectomy is performed to 
induce hepatocyte proliferation (Jaenisch and Hoffman, 1979). It was also shown that 
transgenic proviral sequences were not constitutively transcribed in hepatocytes 
(Stuhlmann et al, 1984; derPutten et al, 1985). This pessimistic outlook has been 
dissipated by studies which demonstrate efficient gene transfer into primary hepatocytes 
using retroviral vectors (Ledley et al, 1987a; Peng et al, 1988; Wilson et al, 1988a; 
1988b; Wolff et al, 1987a; 1987b; Anderson et al, 1989). It should be noted that the 
early work of Jaenisch never demonstrated that retroviruses gained access to hepatocytes 
in the lamina and also failed to detect transduction of endothelial cells or fibroblasts 
within the liver which are known to be infectable under appropriate conditions . 
Initial experiments in the laboratory of Dr. Savio Woo used phenylketonuria as a 
I model for a disease which would require liver- specif ic gene replacement. Phenylalanine 
j hydroxylase (PAH) is a liver specific gene which hydroxylates phenylalanine to tyrosine 
and requires a reduced tetrahydrobiopterin cofactor (Scriver et al, 1989). 
Tetrahydrobiopterin is synthesized from GTP primarily by the liver and is maintained in 
I the reduced state by the enzyme dihydropteridine reductase. A recombinant retroviral 
! vector was generated which contained the human gene in the retroviral vector pZIPNEO- 
SV(X) (Cepko et al, 1984). High titer vector (lO^cfu/ml) was obtained and shown to be 
capable of transducing the PAH gene into NIH3T3 cells (Ledley et al , 1986). Although 
Recombinant DNA Research, Volume 14 
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