Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection).

G3139, an anti-Bcl-2 antisense oligomer that binds heparin-binding growth factors and collagen I, alters in vitro endothelial cell growth and tubular morphogenesis.

Purpose: We examined the effects of G3139 on the interaction of heparin-binding proteins [e.g., fibroblast growth factor 2 (FGF2) and collagen I] with endothelial cells.

Angiogenesis alteration by defibrotide: implications for its mechanism of action in severe hepatic veno-occlusive disease.

Defibrotide (DF) is a mixture of porcine-derived single-stranded phosphodiester oligonucleotides (9-80-mer; average, 50-mer) that has been successfully used to treat severe hepatic veno-occlusive disease (sVOD) with multiorgan failure (MOF) in patients who have received cytotoxic chemotherapy in preparation for bone marrow transplantation. However, its mechanism of action is unknown. Herein, we show that DF and phosphodiester oligonucleotides can bind to heparin-binding proteins (eg, basic fibroblast growth factor [bFGF] but not vascular endothelial growth factor [VEGF] 165) with low nanomolar affinity.

Delivery of G3139 using releasable PEG-linkers: impact on pharmacokinetic profile and anti-tumor efficacy.

In order to overcome the problems of enzymatic degradation and short plasma half life, which can limit the delivery of antisense oligonucleotides, and the potential immuno-stimulatory effects of CpG motifs, we utilized a polyethylene glycol (PEG) technology that employed various releasable linkers (rPEG). 5'-20 kDa-PEGylation of an anti-Bcl-2 5'-aminoalkyl-oligonucleotide with the same sequence as G3139 (Compound 1) did not alter its binding to the heparin-binding protein bFGF, nor the release of cytochrome c from isolated mitochondria treated with the conjugates. However, in 518A2 melanoma cells in vitro, PEGylation resulted in greatly diminished cellular uptake.

Comparison of d-g3139 and its enantiomer L-g3139 in melanoma cells demonstrates minimal in vitro but dramatic in vivo chiral dependency.

G3139 (Genasense), an 18mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 messenger RNA (mRNA), downregulates Bcl-2 protein and mRNA expression in many cell lines. However, both the in vitro and in vivo mechanisms of action of G3139 are still uncertain. The isosequential L-deoxyribose enantiomer L-G3139, which does not downregulate Bcl-2 expression, was synthesized to study the role of the Bcl-2 protein in melanoma cells.