Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes.

Abstract

We have combined a receptor-mediated DNA delivery system with the endosomal lysis ability of adenovirus and shown that DNA can be delivered into primary hepatocytes, resulting in a high level of gene expression. When asialoorosomucoid conjugated with poly(L-lysine) was used to deliver the Escherichia coli ,t-galactosidase gene into primary hepatocytes through binding with the hepatic asialoglycoprotein receptor, only a low level of p-galactosidase was detectable, with less than 0.1% of the hepatocytes being transfected. This level of activity can be greatly enhanced by the cointernalization of the DNA-protein complex with a replication-defective adenovirus, resulting in 100% of the hepatocytes staining blue with 5-bromo-4-chloro-3-indolyl ,B-D-galactoside. Quantitative analysis of g8-galactosidase expression also showed a 1000-fold enhancement of activity. To test the applicability of this DNA delivery system for the correction of phenylketonuria, a metabolic disorder that causes severe mental retardation in children, we have delivered the human phenylalanine hydroxylase (PAH) gene to hepatocytes derived from a PAH-deficient mouse strain and demonstrated complete reconstitution of enzymatic activity. This method shows great promise for efficient gene delivery to the liver for correction of hepatic disorders.The focus of research in gene therapy during the past 3-5 years has been on using both viral (1-5) and nonviral (6-8) methods of delivery that rely on surgical procedures to facilitate gene delivery and expression (1,2). Future therapies will rely on the development of DNA delivery systems with target organ specificity that do not require major surgical procedures. The creation of direct DNA delivery systems by receptor-mediated endocytosis using DNA-protein complexes has contributed to the formation of an attractive gene delivery system. The asialoglycoprotein receptor has been used in targeting DNA to HepG2 cells in vitro (9, 10) and liver cells in vivo (11)(12)(13)(14). The transferrin receptor has been used to deliver DNA to many cell types in vitro (15)(16)(17). The ability to form these DNA-protein complexes relies on formation of toroid structures that are 100 nm in diameter (18). Although DNA can be delivered by this method, it is inefficient without the use of agents that affect the endosome/lysosome pathway, such as chloroquine (17). Replication-deficient adenovirus has recently been found to have the ability to enhance the transfer of transferrin-DNA complexes into HeLa cells by 2000-fold (19), as it promotes endosome lysis.The liver is an excellent target organ for gene therapy, as there are a multitude of human metabolic disorders secondary to hepatic enzyme deficiencies. To develop therapies for these disorders and to test the ability of DNA-protein complexes to deliver genes to primary hepatocytes, we have synthesized asialoglycoprotein-poly(L-lysine) conjugates for DNA-protein complex formation. Quantitative enhancement of DNA delivery and functional expression in hepatocytes were achi...

Enhancement of calcium phosphate-mediated transfection by inclusion of adenovirus in coprecipitates

“…As indicated above, adenovirus has been incorporated into other gene transfer vectors, including cationic lipids and receptortargeted polylysine complexes. [5][6][7][8]10,12,14 In these cases adenovirus is also thought to facilitate escape of the plasmid from the endosome.…”

“…A number of methods have been developed to facilitate plasmid-mediated gene transfer, including direct injection, electroporation, and incorporation of plasmid in complexes with cationic lipids, polylysine, and other molecules. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] The use of calcium phosphate (CaPi) to coprecipitate DNA was one of the first methods used for DNA transfer to cells. 16 CaPi, as well as cationic lipids and other cationic polymers, condense the DNA and facilitate its uptake into the cell.…”

“…A similar strategy has been described in which adenovirus was included in complexes of DNA plus a cationic molecule (polylysine or cationic lipids), and in some cases a targeting moiety. [5][6][7][8]10,12,14 In such strategies, the adenovirus was either covalently linked to the cationic molecule or included in the complex via electrostatic interactions. Including adenovirus in the complex by either method markedly improved delivery and expression of plasmid DNA.…”

State-of-the-art 2003 on PKU gene therapy

“…The transduction of cells by such DNA/protein complexes in vitro was greatly increased by the coadministration of a replication-defective adenovirus and reached an efficiency of 100% [30]. Although the low levels of PAH activity were reconstituted to nearly normal ranges, this method has produced very small portions of transduced hepatocytes in vivo [31].…”

Non-viral approaches to gene therapy

“…Non-viral approaches to gene therapy Cotten and addition of adenovirus particles, which are naturally endosomolytic [30-321, enhances gene delivery [33][34][35] and is particularly effective when the DNA to be delivered is linked directly to the exterior of the adenovirus particle [36]. Other, more inert, viruses can also be used for this purpose: the chicken adenovirus (CELO virus) is naturally defective in the mammalian cell and provides a potent endosome disruption activity [37].…”