title: Targeted delivery of antisense oligonucleotides by molecular conjugates.
abstract: Antisense oligonucleotides efficiently inhibit gene expression in vitro; however, the successful therapeutic application of this technology in vivo will require the development of improved delivery systems. In this report we describe a technique that efficiently delivers antisense oligonucleotides into cells using molecular conjugates. This technique, which was initially developed for the delivery of eukaryotic genes, is based on the construction of DNA-protein complexes that are recognized by the liver-specific asialoglycoprotein receptor. Binding of poly(L-lysine)-asialoorosomucoid (AsOR) protein conjugates with phosphorothioate antisense oligonucleotides to chloramphenicol acetyltransferase (CAT) led to the formation of 50- to 150-nm toroids. Exposure of the antisense molecular complexes (3 microM oligonucleotide) to NIH 3T3 cells genetically modified to express both the AsOR receptor and CAT, inhibited CAT expression by 54%, which was completely blocked by excess AsOR. Equivalent inhibition of CAT activity with purified oligonucleotide alone was observed at a 30 microM concentration. Furthermore, examination of the cells using indirect immunofluorescence for the presence of CAT protein showed 28% of cells exposed to the molecular conjugates lacked any detectable CAT enzyme. Cells exposed to oligonucleotide alone showed a highly variable staining pattern, and only a few of the cells were completely void of CAT protein. Together these data demonstrate that molecular conjugates provide a highly specific and efficient system for the delivery of antisense oligonucleotides.
authors.affiliation: Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor 48109.
authors.forename: B A
authors.initials: BA
authors.lastname: Bunnell
authors.forename: F K
authors.initials: FK
authors.lastname: Askari
authors.forename: J M
authors.initials: JM
authors.lastname: Wilson
mesh_terms: D016475:3T3 Cells; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004337:Drug Carriers; D007158:Immunologic Techniques; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000235:genetics; D009961:Orosomucoid / Q000031:analogs & derivatives; D011107:Polylysine / Q000031:analogs & derivatives; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D013873:Thionucleotides / Q000737:chemistry
chemical_list: D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D004337:Drug Carriers; D016376:Oligonucleotides, Antisense; D009961:Orosomucoid; D011971:Receptors, Immunologic; D013873:Thionucleotides; C057326:asialoorosomucoid-polylysine conjugate; D011107:Polylysine; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase