Receptor-mediated delivery of foreign genes to hepatocytes
Introduction
Parenchymal liver cells, hepatocytes, are useful target cells for gene delivery because they can perform a host of post-translational modifications that may be required for the activity of certain gene products [1]. The cells are highly active metabolically. The liver has a rich blood supply that can be useful for the delivery of genes to the liver, and also, for the distribution of gene products from the liver to the systemic circulation. Finally, mammalian hepatocytes are the only cells that possess large numbers of high affinity cell-surface receptors that can bind asialoglycoproteins. Asialoglycoproteins are a class of glycoproteins that possess clustered galactose residues that are required for recognition and binding by asialoglycoprotein receptors. This receptor system can not only bind galactose-containing ligands, but can internalize them within membrane-bound vesicles or endosomes [2], Fig. 1. Because of the highly selective presence of these receptors on hepatocytes, agents can be targeted specifically to those cells using asialoglycoproteins as carriers [3].
Section snippets
A carrier system for targeting DNA to hepatocytes
Targeted delivery of DNA to liver cells via asialoglycoprotein receptors was accomplished using a protein conjugate consisting of: a cell targeting component composed of an asialoglycoprotein ligand covalently bound to a DNA binding component consisting of a polymer containing multiple positive charges (polylysine) [4].
Targeted gene delivery in vitro
Initial studies testing this carrier system were carried out in vitro. A plasmid carrying the chloramphenicol acetyltransferase gene (CAT) gene as a marker was complexed to the conjugate [5]and given to two cell lines under identical conditions: an asialoglycoprotein (AsG) receptor (+) cell line, and an AsG receptor (−) cell line. After incubation, no CAT activity could be demonstrated in AsG receptor (−) cells. However, CAT activity was detected in complex-treated AsG receptor (+) cells. In
Targeted gene delivery in vivo
Subsequent experiments were performed to determine whether the asialoglycoprotein-based DNA delivery system could target DNA to hepatocytes in vivo. 32P-labeled DNA alone or as a complex with AsOR-PL conjugate, were injected intravenously into groups of rats. In rats treated with DNA alone, 55% of the radioactivity, was found still circulating in the blood while 17% was detected in the liver 10 min after injection. However, in rats injected with DNA in the form of a complex, 85% of the counts
Transient gene expression in animal models of inherited metabolic disorders
In collaboration with James Wilson, a plasmid containing a cDNA for the human LDL receptor was complexed and injected intravenously into Watanabe rabbits to test the feasibility of targeting the gene for the low density lipoprotein (LDL) receptor in a model for familial hypercholesterolemia. Livers removed 10 min after injection, were found to have approximately 1000 copies of plasmid per cell by Southern blot. However, by 48 h plasmid DNA was less than 0.1 copies/cell. To assess the
Prolonging the duration of targeted gene expression
The initial strategy to prolong the duration of targeted gene expression was based on the possibility that stimulation of hepatocyte replication might enhance the likelihood of integration of the foreign gene into the genome of recipient cells. Although in normal adult liver, there are few dividing hepatocytes, hepatocytes can be induced to replicate in response to injury, e.g. partial hepatectomy [9]. Rats were injected with complexed DNA and then subjected to two-thirds partial hepatectomies.
The mechanism of persistence of targeted gene expression
Experiments were performed to determine the mechanism by which persistent gene expression following partial hepatectomy had occurred. Southern blot analysis of DNA extracted from liver as a function of time after injection and partial hepatectomy showed that 10 min after partial hepatectomy, levels of transgene DNA were 1,000–10,000 copies/cell. The predominant form of this DNA was episomal based on analyses of uncut DNA, or DNA cut with a single cutter endonuclease. Furthermore, the plasmid
Effects of endosomolytic agents
In order to avoid lysosomal degradation, agents that could lyse endosomal vesicles were studied. For example, adenoviruses enter cells by binding to plasma membrane of the host cell through interaction with viral fibers that project from the capsid [19]. Binding of the virus triggers an endocytotic process in which the viral particles are internalized within endosomal vesicles [20]. Acidification of endosomes results in a conformational change of the capsid proteins that disrupts the endosomal
Targeted delivery of single stranded (antisense) DNA
Because the receptor-mediated DNA targeting system was based on electrostatic interactions between the carrier and nucleic acid, it was conceivable that single-stranded DNA in the form of antisense oligomers could be targeted in the same manner as had been demonstrated for double-stranded DNA. Thus, antisense DNA could potentially be targeted in a similar fashion. Because of the inhibition of the synthesis of specific proteins based on hybridization of antisense to target mRNA, it was
Conclusions
Receptor-mediated carrier systems based on the targeting of asialoglycoproteins to highly selective receptors on parenchymal liver cells, hepatocytes, can be used to introduce new gene expression. In addition, the system can introduce antisense nucleotides to suppress endogenous gene expression [27].
Acknowledgements
This work was supported in part by grants from: NIH DK 42182 (GYW) and TargeTech, Inc./Immune Response Corp. (CHW). (GYW and CHW hold equity in the Immune Response Corp.)
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