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Advantages and disadvantages of viral vectors and nonviral vectors for gene delivery to digestive organs are reviewed. Advances in systems for the introduction of new gene expression are described, including self-deleting retroviral transfer vectors, chimeric viruses and chimeric oligonucleotides. Systems for inhibition of gene expression are discussed, including antisense oligonucleotides, ribozymes and dominant-negative genes.

Gene therapy toward the digestive organs has made substantial progress. The strategies applied include ex vivo and in vivo delivery. The ex vivo pathway does not require a tissue-specific vector while the in vivo pathway is advantageous for tissues not obtainable or hard to culture. The specific vectors of gene delivery for the liver and gastrointestinal tract include (1) viral vectors: retrovirus, adenovirus, and adeno-associated virus are the three most common currently used. (2) liposomes: when cationic substances are incorporated, DNA can be coated on the surface and carried into cells. (3) receptor-mediated gene therapy: natural and physiological and tissue-specific ligands were incorporated and modified to carry foreign DNA into the cells. They are tissue-specific and relatively nontoxic. The innovations focus on the prolongation of gene expression and a more efficient gene delivery.

There has been much progress in our understanding of molecular mechanisms in the pathogenesis of inherited metabolic disorders. In addition, powerful new molecular techniques have made possible phenotypic alterations by delivery of foreign genes to target cells. As a result, concepts and methods that would have been considered purely science fiction 10 years ago can now be found in human clinical trials engaged in the treatment of these disorders. In this review, we have attempted to provide an introduction and survey of the topic of gene therapy, with specific examples of laboratory and clinical achievements to date, and highlights on potentials for applications in digestive diseases.

A plasmid (palb3) was constructed containing the structural gene for human serum albumin driven by mouse albumin enhancer-rat albumin promoter elements. Using an asialoglycoprotein-polycation conjugate consisting of asialoorosomucoid coupled to poly-L-lysine, a soluble DNA complex was formed that was capable of targeting specifically to hepatocytes via asialoglycoprotein receptors present on these cells. Groups of Nagase analbuminemic rats were injected with complexed DNA or controls, followed by two-thirds partial hepatectomy to stimulate hepatocyte replication. Using a cDNA probe for the human albumin structural gene, hybridizable sequences were detected in analbuminemic rats treated with complex as determined by Southern blot analysis. Two weeks post-injection, the targeted DNA was found to exist primarily in plasmid form with an average copy number of 1000/diploid cell. Human albumin mRNA was detected by dot-blot hybridization with a specific oligonucleotide cDNA probe and confirmed by RNase protection assay using a vector-specific probe. Circulating human albumin was detected in the serum of palb3-treated Nagase analbuminemic rats by Western blots using an antibody specific for human serum albumin. A time course demonstrated that circulating human albumin was not detectable 24 h after injection, but became measurable at a level of 0.05 micrograms/ml within 48 h and increased in concentration to a maximum of 34 micrograms/ml by 2 weeks post-injection. This level of expression remained stable through 4 weeks after injection and partial hepatectomy.

Introduction of foreign genes into mammalian cells in vitro has been accomplished previously by a variety of methods. The few techniques that have been developed for transfection of mammalian cells in vivo, are technically difficult or lack cell specificity. We have developed a soluble, targetable DNA carrier system consisting of an asialoglycoprotein covalently coupled to a polycation. The strategy was based on: 1) the presence of unique receptors on hepatocytes which internalize galactose-terminal (asialo-)glycoproteins; 2) polycations can bind DNA in a non-covalent, non-damaging interaction. Using chloramphenicol acetyltransferase (CAT) as a marker gene, specific delivery and expression of CAT was demonstrated in vitro using asialoglycoprotein receptor (+) and (-) cell lines. Intravenous injection of conjugate-DNA complexes in rats resulted in detection of CAT DNA sequences in liver 10 min later by dot blots with a CAT cDNA probe. CAT enzyme activity 24 hrs later was found specifically in liver but no other tissues or control livers. Targeted hepatic CAT expression was transient, maximal at 24 hrs but declined to barely detectable levels by 96 hrs. Persistent foreign gene expression was achieved by injection of DNA complex followed by 67% partial hepatectomy. High levels of hepatic CAT activity were detected through 11 weeks post-hepatectomy. The data indicate that a targetable gene delivery system can permit in vivo expression of an exogenous gene after simple intravenous injection. The foreign gene expression can be enhanced and made to persist by induction of hepatocyte replication.