Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR-21 with enhanced in vivo therapeutic potency.

The design of modified oligonucleotides that combine in one molecule several therapeutically beneficial properties still poses a major challenge. Recently a new type of modified mesyl phosphoramidate (or µ-) oligonucleotide was described that demonstrates high affinity to RNA, exceptional nuclease resistance, efficient recruitment of RNase H, and potent inhibition of key carcinogenesis processes in vitro. Herein, using a xenograft mouse tumor model, it was demonstrated that microRNA miR-21-targeted µ-oligonucleotides administered in complex with folate-containing liposomes dramatically inhibit primary tumor growth via long-term down-regulation of miR-21 in tumors and increase in biosynthesis of miR-21-regulated tumor suppressor proteins. This antitumoral effect is superior to the effect of the corresponding phosphorothioate. Peritumoral administration of µ-oligonucleotide results in its rapid distribution and efficient accumulation in the tumor. Blood biochemistry and morphometric studies of internal organs revealed no pronounced toxicity of µ-oligonucleotides. This new oligonucleotide class provides a powerful tool for antisense technology.

Catalytic Knockdown of miR-21 by Artificial Ribonuclease: Biological Performance in Tumor Model.

Control of the expression of oncogenic small non-coding RNAs, notably microRNAs (miRNAs), is an attractive therapeutic approach. We report a design platform for catalytic knockdown of miRNA targets with artificial, sequence-specific ribonucleases. miRNases comprise a peptide [(LeuArg)2Gly]2 capable of RNA cleavage conjugated to the miRNA-targeted oligodeoxyribonucleotide, which becomes nuclease-resistant within the conjugate design, without resort to chemically modified nucleotides. Our data presented here showed for the first time a truly catalytic character of our miR-21-miRNase and its ability to cleave miR-21 in a multiple catalytic turnover mode. We demonstrate that miRNase targeted to miR-21 (miR-21-miRNase) knocked down malignant behavior of tumor cells, including induction of apoptosis, inhibition of cell invasiveness, and retardation of tumor growth, which persisted on transplantation into mice of tumor cells treated once with miR-21-miRNase. Crucially, we discover that the high biological activity of miR-21-miRNase can be directly related not only to its truly catalytic sequence-specific cleavage of miRNA but also to its ability to recruit the non-sequence specific RNase H found in most cells to elevate catalytic turnover further. miR-21-miRNase worked synergistically even with low levels of RNase H. Estimated degradation in the presence of RNase H exceeded 103 miRNA target molecules per hour for each miR-21-miRNase molecule, which provides the potency to minimize delivery requirements to a few molecules per cell. In contrast to the comparatively high doses required for the simple steric block of antisense oligonucleotides, truly catalytic inactivation of miRNA offers more effective, irreversible, and persistent suppression of many copy target sequences. miRNase design can be readily adapted to target other pathogenic microRNAs overexpressed in many disease states.

miRNases: Novel peptide-oligonucleotide bioconjugates that silence miR-21 in lymphosarcoma cells.

MicroRNAs (miRNAs) are active regulators in malignant growth and constitute potential targets for anticancer therapy. Consequently, considerable effort has focused on identifying effective ways to modulate aberrant miRNA expression. Here we introduce and assess a novel type of chemically engineered biomaterial capable of cleaving specific miRNA sequences, i.e. miRNA-specific artificial ribonucleases (hereafter 'miRNase'). The miRNase template presented here consists of the catalytic peptide Acetyl-[(LeuArg)2Gly]2 covalently attached to a miRNA-targeting oligonucleotide, which can be linear or hairpin. The peptide C-terminus is conjugated to an aminohexyl linker located at either the 3'- or 5'-end of the oligonucleotide. The cleavage efficacy, structural aspects of cleavage and biological relevance of a set of these designed miRNases was assayed with respect to highly oncogenic miR-21. Several miRNases demonstrated effective site-selective cleavage of miR-21 exclusively at G-X bonds. One of the most efficient miRNase was shown to specifically inhibit miR-21 in lymphosarcoma cells and lead to a reduction in their proliferative activity. This report provides the first experimental evidence that metallo-independent peptide-oligonucleotide chemical ribonucleases are able to effectively and selectively down-regulate oncogenic miRNA in tumour cells, thus suggesting their potential in development of novel therapeutics aimed at overcoming overexpression of disease-related miRNAs.