Biochemical and functional analysis of an adenovirus-based ligand complex for gene transfer.
Ligand-mediated approaches to gene transfer offer an alternative to viral vectors for both in vivo and in vitro applications. Although a significant percentage of the plasmid-based DNA complex is lost to lysosomal degradation following receptor-mediated endocytosis, simultaneous infection with adenovirus has been shown to increase the level of transgene expression [Curiel, Agarwal, Wagner and Cotten (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8850-8854; Wagner, Zatloukal, Cotten, Kirlappos, Mechtler, Curiel and Birnstiel (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6099-6103]. In this study we describe an adenovirus-based ligand complex where the plasmid DNA, polycation-ligand conjugate and adenovirus are contained within a single particle structure. At the core of the transfection particle is a replication-defective recombinant adenovirus encoding a cDNA minigene for human placenta alkaline phosphatase that was chemically modified with poly(L-lysine) (Ad-pLys). Electron microscopy of an adenovirus-based ligand complex formed by successively adding plasmid DNA and an asialo-orosomucoid-poly(L-lysine) conjugate to Ad-pLys revealed structures that appeared as intact viral particles coated with a dense biomolecular layer. Adenovirus-based ligand complexes containing either a luciferase or beta-galactosidase reporter plasmid were shown to efficiently deliver the plasmid transgene to cells that express the hepatic asialoglycoprotein receptor. Furthermore, the poly(L-lysine) modification greatly reduced the infectivity potential of the virus without causing a concomitant loss of augmented gene transfer. As an alternative to infectious virions, incomplete products of viral assembly were also considered as a source for endosomalytic activity. However, these defective virions were unable to significantly enhance plasmid transgene delivery.
Fisher
K J
KJ
Department of Molecular and Cellular Engineering, University of Pennsylvania Medical Center, Philadelphia.
Wilson
J M
JM
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism; D002213:Capsid / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000378:metabolism; D010957:Plasmids; D011107:Polylysine; D011956:Receptors, Cell Surface / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D008024:Ligands; D009961:Orosomucoid; D011956:Receptors, Cell Surface; C017030:asialoorosomucoid; D011107:Polylysine; D000469:Alkaline Phosphatase