Definition and identification of small RNA sponges: Focus on miRNA sequestration.

Targeting RNAs appears as an important opportunity to modulate biological processes. Here, we overviewed critical parameters implied in RNAs competition to bind small RNAs. These competitions influence small RNA availability and thereby gene expression and cell fate. We focused on the ability of RNAs to sequester small RNA, mainly the microRNAs (miRNAs) and proposed experimental workflows to demonstrate the existence and activity of RNA-sponge. From this basic science, we detailed tailored oligonucleotides, developed to challenge the binding of small RNA. In vitro and in vivo, these tailored oligonucleotides efficiently restore small RNA activity by preventing their sequestration on RNA-sponges.

Antisense oligonucleotides containing conformationally constrained 2',4'-(N-methoxy)aminomethylene and 2',4'-aminooxymethylene and 2'-O,4'-C-aminomethylene bridged nucleoside analogues show improved potency in animal models.

To identify chemistries and strategies to improve the potency of MOE second generation ASOs, we have evaluated gapmer antisense oligonucleotides containing BNAs having N-O bonds. These modifications include N-MeO-amino BNA, N-Me-aminooxy BNA, 2',4'-BNA(NC)[NMe], and 2',4'-BNA(NC) bridged nucleoside analogues. These modifications provided increased thermal stability and improved in vitro activity compared to the corresponding ASO containing the MOE modification. Additionally, ASOs containing N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] modifications showed improved in vivo activity (>5-fold) compared to MOE ASO. Importantly, toxicity parameters, such as AST, ALT, liver, kidney, and body weights, were found to be normal for N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] ASO treated animals. The data generated in these experiments suggest that N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] are useful modifications for applications in both antisense and other oligonucleotide based drug discovery efforts.

Comparing in vitro and in vivo activity of 2'-O-[2-(methylamino)-2-oxoethyl]- and 2'-O-methoxyethyl-modified antisense oligonucleotides.

A number of 2'- O-modified antisense oligonucleotides have been reported for their potential use in oligonucleotide-based therapeutics. To date, most of the in vivo data has been generated for 2'-O-MOE (2'-O-methoxyethyl)- and 2'-O-Me (2'-O-methyl)-modified ASOs (antisense oligonucleotides). We now report the synthesis and biological activity of another 2'-O-modification, namely 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA). This modification resulted in an increase in the affinity of antisense oligonucleotides to complementary RNA similar to 2'-O-MOE-modified ASOs as compared to first-generation antisense oligodeoxynucleotides. The ASO modified with 2'-O-NMA reduced expression of PTEN mRNA in vitro and in vivo in a dose-dependent manner similar to 2'-O-MOE modified ASO. Importantly, toxicity parameters such as AST, ALT, organ weights, and body weights were found to be normal similar to 2'-O-MOE ASO-treated animal models. The data generated in these experiments suggest that 2'-O-NMA is a useful modification for potential application in both antisense and other oligonucleotide-based drug discovery efforts.

Radiolabeling small RNA with technetium-99m for visualizing cellular delivery and mouse biodistribution.

To develop a noninvasive direct method for the in vivo tracking of small interfering RNA (siRNA) used in RNA interference, two 18-nucleotide oligoribonucleotides were radiolabeled with technetium-99m ((99m)Tc-RNA). The ability of (99m)Tc-RNA to track delivery was tested in cultured cells and living mice. The cellular delivery of (99m)Tc-RNAs could be quantified by gamma counting and could be visualized by microautoradiography. Radiolabeled RNAs can be efficiently delivered into cells by reaching up to 3x10(5) molecules of small RNAs per cell. Moreover, RNAs were internalized with homogeneous distribution throughout the cytoplasm and nucleus. In tumor-bearing mice, whole-body images and biodistribution studies showed that (99m)Tc-RNAs were delivered to almost all tissues after intravenous injection. The imaging of living animals allowed noninvasive and longitudinal monitoring of the in vivo delivery of these small RNAs. In conclusion, using (99m)Tc radiolabeling, the delivery of small RNAs could be measured quantitatively in cultured cells and could be noninvasively visualized in living animals using a gamma camera. The results of this study could open up a new approach for measuring the in vivo delivery of small RNAs that might further facilitate the development of siRNAs as targeted therapies.

Chemistry and biochemistry of 2',5'-oligoadenylate-based antisense strategy.

This review describes the application of a natural defense mechanism to develop effective agents for the post-transcriptional control of gene expression. 2-5A is a unique 2',5'-phosphodiester bond linked oligoadenylate, (pp)p5'A2'(p5'A)(n), that is elaborated in virus-infected interferon-treated cells. The 2-5A system is an RNA degradation pathway that is an important mechanistic component of interferon's action against certain viruses. It may also play a role in the anticellular effects of interferon and in general RNA decay. A major player in the 2-5A-system is the latent and constitutive 2-5A-dependent ribonuclease (RNase L) which upon activation by 2-5A, degrades RNA. This RNase L enzyme can be recruited for antisense therapeutics by linking it to an appropriate oligonucleotide targeted to a chosen RNA. Syntheses of 2-5A, its analogues, 2-5A-antisense, and its modifications are detailed herein. Applications of 2-5A-antisense to particular targets such as HIV, PKR, chronic myelogenous leukemia, telomerase, and respiratory syncytical virus are described.

Controlling gene expression with 2-5A antisense.

Recent work has demonstrated that the activity of a ubiquitous cellular enzyme, ribonuclease L (RNase L), can be harnessed to cleave targeted RNA species. Activation of RNase L is dependent on the presence of 2',5'-linked oligoadenylates (2-5A), usually produced by cells infected with viruses. By conjugating synthetic 2-5A to specific antisense compounds, it is now possible to selectively degrade RNAs in an RNase L-dependent manner, thereby providing an alternative to RNase H-dependent approaches. In this summary, we provide an updated description of the synthesis procedure for constructing these chimeric 2-5A antisense molecules. Examples of successful applications of the 2-5A antisense strategy are described, along with some of the procedures involved in those studies. Several methods are also provided for optimizing compound uptake and analyzing their effects on cells. Finally, we discuss the current body of evidence that supports the contention that RNase L is indeed the primary mediator of 2-5A antisense effects and the possible implications that this has on the future of this therapeutic approach.