Dataset Information

Efficient cationic lipid-mediated delivery of antisense oligonucleotides into eukaryotic cells: down-regulation of the corticotropin-releasing factor receptor.

ABSTRACT: Oligonucleotides (ODNs) can be employed as effective gene-specific regulators. However, before ODNs can reach their targets, several physical barriers have to be overcome, as although ODNs may pass cell membranes, most become sequestered in endocytic compartments. Accordingly, sophisticated strategies are required for efficient delivery. Here we have employed a pyridinium-based synthetic amphiphile, called SAINT-2, which carries ODNs into cells in a highly efficient, essentially non-toxic and serum-insensitive manner. Intracellular delivery was examined by monitoring the trafficking of fluorescent ODNs and lipid, and by measuring the effect of specific antisense ODNs on target mRNA and protein levels of the receptor for the neuropeptide corticotropin-releasing factor (CRF-R), expressed in Chinese hamster ovary cells. ODN delivery is independent of lipoplex size, and fluorescently tagged ODNs readily acquire access to the nucleus, whereas the carrier itself remains sequestered in the endosomal-lysosomal pathway. While the release is independent of the presence of serum, it is not observed when serum proteins gain access within the lipoplex, and which likely stabilizes the lipoplex membrane. We propose that the amphiphile-dependent aggregate structure governs complex dissociation, and hence, the biological efficiency of ODNS: We demonstrate an essentially non-toxic and effective antisense-specific down-regulation of the CRF-R, both at the mRNA and protein level.

SUBMITTER: Shi F

PROVIDER: S-EPMC55445 | biostudies-literature | 2001 May

REPOSITORIES: biostudies-literature

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Efficient cationic lipid-mediated delivery of antisense oligonucleotides into eukaryotic cells: down-regulation of the corticotropin-releasing factor receptor.

Shi F F Nomden A A Oberle V V Engberts J B JB Hoekstra D D

Nucleic acids research 20010501 10

Oligonucleotides (ODNs) can be employed as effective gene-specific regulators. However, before ODNs can reach their targets, several physical barriers have to be overcome, as although ODNs may pass cell membranes, most become sequestered in endocytic compartments. Accordingly, sophisticated strategies are required for efficient delivery. Here we have employed a pyridinium-based synthetic amphiphile, called SAINT-2, which carries ODNs into cells in a highly efficient, essentially non-toxic and se ...[more]

PMID: 11353077

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Project description:Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease affecting the upper and lower motor neurons in the motor cortex and spinal cord. Abnormal accumulation of mutant superoxide dismutase I (SOD1) in motor neurons is a pathological hallmark of some forms of the disease. We have shown that the orderly progression of the disease may be explained by misfolded SOD1 cell-to-cell propagation, which is reliant upon its active endogenous synthesis. Reducing the levels of SOD1 is therefore a promising therapeutic approach. Antisense oligonucleotides (ASOs) can efficiently silence proteins with gain-of-function mutations. However, naked ASOs have a short circulation half-life and are unable to cross the blood brain barrier (BBB) warranting the use of a drug carrier for effective delivery. In this study, calcium phosphate lipid coated nanoparticles (CaP-lipid NPs) were developed for delivery of SOD1 ASO to motor neurons. The most promising nanoparticle formulation (Ca/P ratio of 100:1), had a uniform spherical core-shell morphology with an average size of 30 nm, and surface charge (?-potential) of -4.86 mV. The encapsulation efficiency of ASO was 48% and stability studies found the particle to be stable over a period of 20 days. In vitro experiments demonstrated that the negatively charged ASO-loaded CaP-lipid NPs could effectively deliver SOD1-targeted ASO into a mouse motor neuron-like cell line (NSC-34) through endocytosis and significantly down-regulated SOD1 expression in HEK293 cells. The CaP-lipid NPs exhibited a pH-dependant dissociation, suggesting that that the acidification of lysosomes is the likely mechanism responsible for facilitating intracellular ASO release. To demonstrate tissue specific delivery and localization of these NPs we performed in vivo microinjections into zebrafish. Successful delivery of these NPs was confirmed for the zebrafish brain, the blood stream, and the spinal cord. These results suggest that CaP-lipid NPs could be an effective and safe delivery system for the improved delivery of SOD1 ASOs to motor neurons. Further in vivo evaluation in transgenic mouse models of SOD1 ALS are therefore warranted.

Project description:Synthetic nucleic acids have shown great potential in the treatment of various diseases. Nevertheless, the selective delivery to a target tissue has proved challenging. The coupling of nucleic acids to targeting peptides, proteins, and antibodies has been explored as an approach for their selective tissue delivery. Nevertheless, the preparation of covalently coupled peptides and proteins that can also undergo intracellular release as well as deliver more than one copy of the nucleic acid has proved challenging. Recently, we have developed a novel method for the rapid noncovalent conjugation of nucleic acids to targeting single chain antibodies (scFv) using chemically self-assembled nanostructures (CSANs). CSANs have been prepared by the self-assembly of two dihydrofolate reductase molecules (DHFR(2)) and a targeting scFv in the presence of bis-methotrexate (bis-MTX). The valency of the nanorings can be tuned from one to eight subunits, depending on the length and composition of the linker between the dihydrofolate reductase molecules. To explore their potential for the therapeutic delivery of nucleic acids as well as the ability to expand the capabilities of CSANs by incorporating smaller cyclic targeting peptides, we prepared DHFR(2) proteins fused through a flexible peptide linker to cyclic-RGD, which targets ?v?3 integrins, and a bis-MTX chemical dimerizer linked to an antisense oligonucleotide (bis-MTX-ASO) that has been shown to silence expression of eukaryotic translation initiation factor 4E (eIF4E). Monomeric and multimeric cRGD-CSANs were then prepared with bis-MTX-ASO and shown to undergo endocytosis in the breast cancer cell line, MDA-MB-231, which overexpresses ?v?3. The bis-MTX-ASO was shown to undergo endosomal escape resulting in the knock down of eIF4E with at least the same efficiency as ASO delivered by oligofectamine. The modularity, flexibility, and common method of conjugation may prove to be a useful general approach for the targeted delivery of ASOs, as well as other nucleic acids to cells.

Dataset Information

Efficient cationic lipid-mediated delivery of antisense oligonucleotides into eukaryotic cells: down-regulation of the corticotropin-releasing factor receptor.

Publications

Efficient cationic lipid-mediated delivery of antisense oligonucleotides into eukaryotic cells: down-regulation of the corticotropin-releasing factor receptor.

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