title: In vitro and in vivo transport and delivery of phosphorothioate oligonucleotides with cationic liposomes.
abstract: A recent strategy in gene therapy has been using antiviral genes that are delivered to uninfected cells, either as RNA or DNA, to provide intracellular protection from human immunodeficiency virus type-1 (HIV-1) infection. Antisense oligonucleotides that are complementary to specific target genes suppress gene expression. A variety of techniques are available to enhance the cellular uptake and pharmacological effectiveness of antisense oligonucleotides, both in vitro and in vivo. We investigated the intracellular and tissue uptake of an oligonucleotide/cationic lipid complex, using a fluorescently labeled oligonucleotide. The antisense oligonucleotide was designed against the HIV-1 gag gene sequence. A T-cell line (MT-4) and PHA-stimulated peripheral blood mononuclear cells (PBMCs) were both infected with HIV-1(NL432) at an MOI of 0.01. One h later, both cultures were washed and treated with medium containing 1 microM antisense oligonucleotide. After a 3-day interval, the HIV-1 antigen expression was monitored by an indirect immunofluorescence assay. At 3 days post infection, we confirmed that p24 antigen production was inhibited by the antisense oligonucleotide/cationic lipid complex at a 1/10 ratio in the PBMCs, using enzyme-linked immunosorbent assay (ELISA). We also confirmed the intracellular existence of the complex by fluorescent microscopy. We investigated different means of transporting the antisense oligonucleotide/cationic lipid complex to mouse tissues by intravenous, intraperitoneal and subcutaneous injections. We observed that the anti-HIV-1 activity of the antisense oligonucleotide/cationic lipid complex was the result of enhanced cellular uptake, both in vitro and in vivo. Therefore, the antisense oligonucleotide/cationic lipid complex is an excellent system for the transport and delivery of genes to target cells, as it is effective both in vitro and in vivo.
authors.affiliation: High Technology Research Center, Chiba Institute of Technology, Chiba, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Miyano-Kurosaki
authors.forename: Jacob Samson
authors.initials: JS
authors.lastname: Barnor
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Takeuchi
authors.forename: Takashi
authors.initials: T
authors.lastname: Owada
authors.forename: Hideki
authors.initials: H
authors.lastname: Nakashima
authors.forename: Naoki
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Matsuzaki
authors.forename: Fumiyuki
authors.initials: F
authors.lastname: Shimada
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takaku
mesh_terms: D000818:Animals; D019380:Anti-HIV Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004337:Drug Carriers; D004353:Drug Evaluation, Preclinical; D005260:Female; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D008081:Liposomes; D051379:Mice; D008807:Mice, Inbred BALB C; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D014018:Tissue Distribution; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D019380:Anti-HIV Agents; D004337:Drug Carriers; D008081:Liposomes; D016376:Oligonucleotides, Antisense