the cultured hepatocytes which will be harvested on day 3 and infused into the portal circulation 
of the patient through an indwelling catheter. 
The patient will be evaluated for engraftment of corrected hepatocytes through a series 
of metabolic studies. Three months after gene therapy, a small amount of liver tissue will be 
harvested by percutaneous biopsy and analyzed for the presence of recombinant derived RNA and 
DNA. 
il. Background 
A. Gene Therapy 
Monumental progress in the areas of molecular biology, virology, gene transfer 
technology, and human genome mapping are providing the foundation for a new era of biomedical 
research called somatic cell gene therapy (20). This innovative approach to therapeutics has 
the potential to prevent, treat, or potentially cure a variety of inherited and acquired diseases. 
Two general strategies are being developed for the treatment of diseases based on somatic 
gene therapy. The most popular approach, which has been used in the first human trials, is 
referred to as ex vivo gene therapy. This is a multistep therapy which involves transplantation 
of genetically modified autologous cells. The specific steps are summarized below. Tissue that 
contains the appropriate target cell is harvested from the patient and the constituent cells are 
isolated, plated in culture, and exposed to recombinant retroviruses which are capable of 
transducing therapeutic genes into a significant proportion of the cells. The genetically modified 
cells are harvested and transplanted into the patient from which they were derived. An 
advantage of this approach is that gene transfer can be accomplished in an efficient and 
controlled manner in vitro and the genetically modified cells can be characterized prior to 
transplantation. Ex vivo gene therapy has several limitations. It is generally an invasive 
therapy requiring two surgical procedures (i.e., tissue harvest and cell transplantation). In 
addition, the efficacy of ex vivo gene therapy will necessarily be dependent upon the capacity and 
performance of the cell transplantation, which in some systems can be limiting. 
An alternative and potentially more effective approach to gene therapy is direct delivery 
of the therapeutic gene to cells in vivo. This would require the development of gene transfer 
substrates capable of targeting to the appropriate cell and internalizing the gene so that it is 
transported to the nucleus and expressed. Techniques of in vivo gene transfer are very early in 
development. 
The development of a specific gene therapy protocol is a complex process that requires 
an understanding of the pathophysiology of the disease as well as an appreciation of the available 
technology. A variety of somatic cells have been considered potential targets for somatic gene 
transfer including hepatocytes (21), hematopoietic stem cells (22), respiratory airway 
epithelial cells (23), lymphocytes (24), endothelial cells (25), skeletal (26) and cardiac 
myocytes (27), fibroblasts (28), keratinocytes (29), and cells of the central nervous system 
(30) . 
An equally wide array of gene transfer substrates are available. Recombinant 
retroviruses are the preferred substrate for transducing genes into dividing cells in vitro 
(31) . Approaches for in vivo gene transfer are early in development and include viral 
[adenoviruses (23), adeno-associated viruses (32), herpes simplex virus (30)] and nonviral 
substrates [liposomes (33,34), and DNA/protein complexes (35)]. 
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