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 (EC50, 1.3 μM). INF7 was therefore selected for coupling to a high affinity ligand for the asialoglycoprotein receptor (ASGPr), K(GalNAc)2, to improve 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.