1. In vitro and in vivo transport and delivery of phosphorothioate oligonucleotides with cationic liposomes.
- Author
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Miyano-Kurosaki N, Barnor JS, Takeuchi H, Owada T, Nakashima H, Yamamoto N, Matsuzaki T, Shimada F, and Takaku H
- Subjects
- Animals, Anti-HIV Agents pharmacokinetics, Biological Transport physiology, Cell Line, Dose-Response Relationship, Drug, Drug Carriers, Drug Evaluation, Preclinical, Female, Gene Expression Regulation, Viral drug effects, HIV-1 genetics, HIV-1 physiology, Humans, In Vitro Techniques, Leukocytes, Mononuclear drug effects, Liposomes, Mice, Mice, Inbred BALB C, Oligonucleotides, Antisense genetics, Oligonucleotides, Antisense pharmacokinetics, Tissue Distribution, Anti-HIV Agents pharmacology, HIV-1 drug effects, Oligonucleotides, Antisense pharmacology, Virus Replication drug effects
- 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.
- Published
- 2004
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