title: Targeted lysosome disruptive elements for improvement of parenchymal liver cell-specific gene delivery.
abstract: The transfection ability of nonviral gene therapy vehicles is generally hampered by untimely lysosomal degradation of internalized DNA. In this study we describe the development of a targeted lysosome disruptive element to facilitate the escape of DNA from the lysosomal compartment, thus enhancing the transfection efficacy, in a cell-specific fashion. Two peptides (INF7 and JTS-1) were tested for their capacity to disrupt liposomes. In contrast to JTS-1, INF7 induced rapid cholesterol-independent leakage (EC(50), 1.3 microm). INF7 was therefore selected for coupling to a high affinity ligand for the asialoglycoprotein receptor (ASGPr), K(GalNAc)(2), to im- prove its uptake by parenchymal liver cells. Although the parent peptide disrupted both cholesterol-rich and -poor liposomes, the conjugate, INF7-K(GalNAc)(2), only induced leakage of cholesterol-poor liposomes. Given that endosomal membranes of eukaryotic cells contain <5% cholesterol, this implies that the conjugate will display a higher selectivity toward endosomal membranes. Although both INF7 and INF7-K(GalNAc)(2) were found to increase the transfection efficiency on polyplex-mediated gene transfer to parenchymal liver cells by 30-fold, only INF7-K(GalNAc)(2) appeared to do so in an ASGPr-specific manner. In mice, INF7-K(GalNAc)(2) was specifically targeted to the liver, whereas INF7 was distributed evenly over various organs. In summary, we have prepared a nontoxic cell-specific lysosome disruptive element that improves gene delivery to parenchymal liver cells via the ASGPr. Its high cell specificity and preference to lyse intracellular membranes make this conjugate a promising lead in hepatocyte-specific drug/gene delivery protocols.
authors.affiliation: Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research and the Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Sabine M W
authors.initials: SM
authors.lastname: Van Rossenberg
authors.forename: Karen M
authors.initials: KM
authors.lastname: Sliedregt-Bol
authors.forename: Nico J
authors.initials: NJ
authors.lastname: Meeuwenoord
authors.forename: Theo J C
authors.initials: TJ
authors.lastname: Van Berkel
authors.forename: Jacques H
authors.initials: JH
authors.lastname: Van Boom
authors.forename: Gijs A
authors.initials: GA
authors.lastname: Van Der Marel
authors.forename: Erik A L
authors.initials: EA
authors.lastname: Biessen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018014:Gene Transfer Techniques; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data