Gene Therapy and Pediatric Liver Disease (2002)

The liver has many critical functions in the metabolism of lipids, carbohydrates, and proteins. The liver synthesizes and recycles most plasma proteins and coagulation factors, clears lipids and toxins from the serum, secretes bile into the intestine, and synthesizes glucose to maintain adequate serum homeostasis (1). Genetic deficiencies that lead to disease in each of these critical liver functions have been identified. Although none of these diseases is very common individually, they are often lethal or crippling at a young age (2). Consequently, early intervention can be critical for preventing serious damage to the liver or to other organs such as the brain. As a group, however, liver-based metabolic diseases are the second most common indication for orthotopic liver transplantation in children (3). Successful gene therapy in liver disorders has been achieved in animal models (4). Introducing the corrective gene has cured rats with jaundice due to a defective glucuronyl/transferase deficiency (5). Mice with tyrosinemia, a disease that causes progressive hepatocellular injury, have been rescued by replacing the defective gene. Mice with phenylketonuria, a disease that results in mental retardation, also have been treated with hepatic gene transfer (6). More common diseases, such as viral infections and hepatitis, are caused by intracellular expression of the viral genome. Gene therapy with virus-blocking antisense constructs has cured ducklings with viral hepatitis. Liver gene therapy also could be used to cure nonliver diseases. Diabetes mellitus, a disease caused by insufficient pancreatic β-cell activity, has been treated in rats by transfection of an insulin construct into the animal's liver cells. Recently, partial metabolic correction was reported when the liver gene for the LDL receptor, key to hepatocyte removal of blood cholesterol, was transfected ex vivo into the defective liver cells of five patients with familial hypercholesterolemia using retroviral vectors and autologous liver cell transplantation (7). Decreased serum cholesterol concentrations were demonstrated, albeit not sufficient to effect a cure. Another disease amenable to gene therapy is hepatocellular carcinoma. Active research is under way to determine optimal gene transfer strategies that either kill malignant cells or regulate their growth. Genetic disorders represent more than 100 different diseases potentially curable with liver gene therapy (8). For many genetic diseases, therapy must be directed specifically at the liver because of the requirement of multiple proteins in specific pathways that are present exclusively or preferentially in the hepatocyte. Hepatic disorders that may be amenable to gene therapy can be grouped into different categories, each of which may require different gene transfer strategies: 1) the absence of a gene's function in the liver may not result in damage to the liver itself, but may cause disease in the brain or other organs (e.g., urea cycle disorders); 2) the absence of gene function can produce progressive liver disease that leads to cirrhosis and death, such as in the progressive familial intrahepatic cholestatic syndromes; 3) mutations of genes involved in organogenesis can result in liver disease (e.g., Alagille syndrome); 4) some mutations result in the production of an altered protein (gain-of-function) that may result in severe liver disease (e.g., ZZ-alpha 1-antitrypsin); and 5) acquired disorders, such as infection and malignancy, also affect the liver and could be amenable to gene therapy. Distinguishing and grouping of these disorders are important because the gene therapy strategy used for each category will be inherently different. AREAS OF EMPHASIS At this stage in gene therapy research, a number of technical hurdles still must be overcome before hepatic gene therapy is a viable treatment option. Nevertheless, new technological breakthroughs, primarily related to vector development, are likely in the near future and could facilitate the development of efficacious clinical trials (9). In the short term, the focus of hepatic gene therapy research will be on treating diseases that have high morbidity and mortality resulting from single enzyme deficiencies (e.g., urea cycle defects). As the Human Genome Project and gene-array technology allow us to identify and delineate genetic susceptibility states (e.g., atherosclerosis, cancer), preventive rather than curative treatments may be possible. DNA testing for numerous conditions and predispositions eventually could replace the current newborn screen, and in the 21st century, gene therapy probably will play a major role in disease prevention and treatment. Direct, in vivo, gene transfer (i.e., intravenous or oral infusion of a vector) and lifelong complete correction of a genetic deficiency should be the ultimate goal for most gene therapy applications. It is unresolved which vectors will be most efficient in achieving this goal. Further study of the regulation of inserted genes is also required. The study of site-directed nucleotide exchange requires emphasis (10). For acquired liver disorders, gene therapy applications should be tailored to each specific disease. Curative treatment of hepatitis, for example, will require efficient (i.e., affecting every hepatocyte) but not permanent expression of a therapeutic molecule, perhaps a ribozyme or antisense nucleotide that can eliminate the viral genome (11). Cancer treatment, however, will require expression of antibody-directed or ligand-directed targeting of toxic or cytolytic fusion proteins or selective expression of an antitumor gene (e.g., wild-type p53 or a cytotoxic gene) that must persist long enough to kill all tumor cells. The recent discovery that liver cells have a stem cell–like potential is encouraging for ex vivo gene therapy strategies and allogeneic cell transplantation as a means of correcting genetic or acquired liver diseases (12–13). Cell transplantation also will continue to have potential for treating nongenetic liver disorders. Actual disease cures may be achieved earlier by using cell therapies than by using in vivo gene therapy methods. This is important for maintaining research funding. Because the gene therapy applications developed in the past decade have not proven curative and because fatal complications related to gene therapy have been reported, funding for future development of these powerful treatment tools may dwindle. RESEARCH GOALS To increase understanding of pediatric liver disease pathophysiology To increase understanding of hepatic cell biology To support development of high-quality gene therapy vectors To determine relevant host–vector interactions To develop liver cell transplantation strategies to correct genetic diseases To develop disease-tailored gene therapy strategies RESEARCH STRATEGIES Pathophysiology of Pediatric Liver Diseases The development of novel therapies for many liver diseases will require a more in-depth understanding of the disease processes specific to each disorder. Such understanding will be achieved by sequencing the genes preferentially expressed in the liver and by determining their functions. In addition, gene-altered expression must be related to specific pathologic states, for example, by using micro-array technology, or by developing appropriate animal models such as gene knockout and transgenic mice. Investigation of gene mutations, expression assays, and structure–function analyses will help clarify the processes involved in genetic and acquired liver diseases. Hepatic Cell Biology Detailed knowledge about the behavior of different liver cell types during normal development, injury, and regeneration is essential for designing rational therapeutic strategies for liver disorders (14). The existence of hepatic progenitor or stem cells may be exploited for ex vivo gene therapy or cell transplantation. Successful in vitro expansion of primary human hepatocytes would have major implications for the treatment of liver disease both in children and in adults. Stellate cells play an important role in the regenerative response and in fibrosis and may be important targets for gene transfer. These experimental studies should involve the modulatory mechanisms for the processes of proliferation, differentiation, and apoptosis of liver cells. Vector Development The main obstacle facing successful gene therapy for many hepatic diseases is the availability of an appropriate vector. The major concerns in developing gene therapy vectors are efficacy and safety. Development of specific vectors also should include tissue-specific targeting and regulable gene expression. Moreover, because the vectors used to date have limitations and the strategies for using gene therapy applications differ, various approaches should be investigated. These approaches should include, but not be limited to, novel vectors, adeno-associated viruses, adenoviruses, retroviruses (including lentiviruses), nonviral DNA-protein complexes, and liposomes. Host–Vector Interactions The vectors used in gene therapy can elicit a robust immunologic response against the vector, the vector-transduced cells, or the transgene product. This immunologic response will become an increasingly important problem as new vectors and different gene products are tested in clinical trials. The mechanisms involved in this immunologic response must be delineated to produce less antigenic vectors and to develop immunomodulatory strategies to blunt the response. Hepatocyte Transplantation Well-controlled clinical trials must be expanded and performed to determine whether cell transplantation is effective in ameliorating acquired and inherited liver diseases. The development of state-of-the-art liver cell banks will allow these trials to be conducted. Additional studies also are necessary to improve the techniques used to cryopreserve primary human hepatocytes and to deliver these hepatocytes to the liver. Critical to the cure of metabolic diseases by liver cell transplantation are clinically relevant methods to confer to transplanted cells a survival advantage in a proliferative environment that can allow significant repopulation of the host liver. Studies are necessary of the mechanisms of cell death during cell transplantation and of the methods to prevent it, thus enhancing engraftment. Gene Therapy Strategies Gene therapy strategies should be tailored to each disease process. Some of the different strategies that can be used are as follows: 1) Gene replacement therapy involves the expression of a protein that is nonfunctional because of a mutation in the host genome. This type of gene therapy will be adequate treatment for many hepatodeficiency states (e.g., inborn errors of metabolism). 2) Gene addition therapy involves the introduction of a novel, exogenous gene product not normally produced in the liver. This type of gene therapy may be appropriate treatment for a number of acquired liver diseases such as fibrosis, cancer, and infections. 3) Gene correction therapy involves the use of oligonucleotides (RNA/DNA chimeras) or viral vectors (e.g., AAV) to change the nucleotide sequence within a chromosomal gene. This type of gene repair has potential advantages over gene replacement (15). For example, any disease (e.g., ZZ-α1-antitrypsin deficiency) in which the mutation results in abnormal function is not correctable by gene replacement. Moreover, repairing a defective gene will result in normal regulation of the expressed gene product in the liver. 4) Gene elimination therapy is another approach for correcting gain-of-function mutations and for removing unwanted nucleic acid sequences that are synthesized secondarily to viral infections. The development of ribozymes and antisense technologies will be critical in gene elimination therapy. PROJECTED TIMETABLE AND FUNDING REQUIREMENTS Gene therapy will most likely be available to treat both genetic and acquired liver diseases, and its use will undoubtedly proliferate during the next several decades as gene therapy technologies continue to improve. The first liver diseases treated with gene therapy will be those with a high benefit–risk ratio. As knowledge in the gene therapy field advances, as is the case with many novel therapies, less severe diseases and conditions related to genetic susceptibility will become amenable to genetic therapies. At this stage, hepatocyte transplantation should be explored as a potential clinical therapy during the next 5 years. Consequently, clinical trials of hepatocyte transplantation should be supported at a level of $400,000 to $500,000 per year. Furthermore, gene therapy vectors will continue to be improved during this decade as our understanding of host–vector interactions grows. The National Institutes of Health and other funding agencies should give priority to supporting the continued development and testing of gene transfer technology. Private industry also will play a major role in the development of efficient and safe gene transfer technological advances. HEALTH AND ECONOMIC OUTCOMES More than 160 children with inherited metabolic diseases undergo orthotopic liver transplantation in the United States each year at a cost that exceeds $40 million. Many more children do not have access to this lifesaving treatment and have chronic, debilitating conditions that require repeated hospitalizations and result in increased long-term healthcare costs. Hepatic gene therapy should dramatically decrease the cost of medical care for these children while improving their quality of life. CONCLUSION Gene therapy is a new and powerful tool being developed by biomedical scientists to correct inherited diseases and to treat diseases caused by viruses and other external factors. Genes are responsible for every characteristic of the human body, including hereditary diseases and predispositions to illness. Gene therapy involves the insertion of curative genes, or of curative DNA segments, either into diseased cells or into nondiseased cells to correct phenotypic abnormalities. The liver not only plays a vital role in almost all aspects of metabolism but it readily incorporates substances and viral vectors for gene therapy. Inherited liver diseases that manifest in childhood are, therefore, an ideal initial focus for hepatic-directed gene therapy research. Much research and development are still necessary before gene therapy becomes an effective treatment for common diseases. Nevertheless, gene therapy promises to be one of the medical breakthroughs of the 21st century, and the liver promises to be the cornerstone.