Position-Dependent Stabilization of DNA/RNA Duplexes by Site-Specific Incorporation of LNA Nucleosides.

The ability of Escherichia coli RNase H1 to hydrolyze structured substrates containing antisense oligonucleotides preannealed to a 47-mer RNA was compared with its ability to hydrolyze unstructured substrates containing antisense oligonucleotides duplexed with 13-mer RNA. These results demonstrate that when antisense oligonucleotides were bound to structured RNA, the resultant duplexes were cleaved at rates significantly slower than when the same oligonucleotides were bound to unstructured oligoribonucleotides. Structured substrates exhibited fewer cleavage sites, and each cleavage site was cleaved less rapidly than in unstructured substrates. Furthermore, the enzymatic activity of E. coli RNase H1 for the structured substrates was most affected when the cleavage sites corresponding to the enzymatically most active sites on the unstructured substrates were blocked in the structured substrates. Molecular modeling suggests that the observed ablation of RNase H activity was due to the steric hindrance of the enzyme by the structured RNA, i.e. steric interference of the phosphate groups on the substrate and/or the binding site of the enzyme. When chimeric oligonucleotides composed of a five-base deoxynucleotide sequence flanked by chemically modified nucleotides were bound to structured RNA, the resultant duplexes were even worse substrates for RNase H. These results offer further insights into the role of antisense-induced RNA structure on RNase H activity and may facilitate the design of effective antisense oligonucleotides.

Peptide-mediated Cell and In Vivo Delivery of Antisense Oligonucleotides and siRNA

Vitravene--another piece in the mosaic.

Oligonucleotide Therapies for the Lung: Ready to Return to the Clinic?

2',4'-methylene bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA; LNA) is a modified nucleic acid that improves the function of antisense oligonucleotide therapeutics. In particular, LNA in the DNA strand increases its binding affinity for the target RNA. Predicting the binding affinities of LNA-containing antisense oligonucleotides and RNA duplexes is useful for designing antisense oligonucleotides. The nearest neighbor parameters may be useful for binding affinity prediction, similar to those for natural nucleic acids. However, the sequence dependence of the thermodynamic stability of DNA/RNA duplexes containing LNA remains unexplored. Therefore, in this study, we evaluated the thermodynamic stabilities of DNA/RNA duplexes containing a single LNA modification in the DNA strand. We found that LNA-stabilized DNA/RNA duplexes averaged -1.5 kcal mol-1. Our findings suggest that the thermodynamic stabilization effect of LNA is sequence-specific.

Electronic Structures of LNA Phosphorothioate Oligonucleotides.

Oligonucleotide Delivery to the Lung: Waiting to Inhale

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Molecular mechanisms of antisense drugs: RNase H.

6 - Antisense Strategies for the Development of Novel Cancer Therapeutics

The inhibition of cellular processes associated with the malignant pheonotype is the goal many oncolytics. In the antisense oligonucleotide (ASO) approach, Watson and Crick base-pairing rules serve as the basis of rational drug design to create single-stranded oligonucleotides that are complementary and bind to RNAs critical for the malignant phenotype. The targets for antisense approaches can be mRNAs that encode for disease related proteins like those that control tumor growth, cell division, or survival. More recently, RNA targets in oncology have been expanded to include microRNAs. A successful oncolytic oligonucleotide must effectively bind and inhibit a target mRNA or miRNA that is critical for the malignant transformation or survival. Target identification presents the same challenge in antisense therapeutics as any other class of oncolytics, but drug design is based the known sequence of the target RNA. The first clinical trials to use antisense oligonucleotides in oncology produced only modest efficacy in clinical trials, but the therapeutic potential of these initial antisense drugs may have been hampered by their low stability and low binding affinity. Advancements in oligonucleotide chemistry have produced oligonucleotides with increased stability and increased affinities. This talk will focus on the use of oligonucleotides with the locked nucleic acid (LNA) modification. In these antisense constructs, some of the nucleotides in the sequence have a modification of the ribose sugar that includes a methylene bridge between the 2’ and 4’ positions. This bridge functions to lock the ribose into a (C3’-endo) configuration: a configuration that is optimized for binding to its cognate nucleotide. LNA-modified oligonucleotides bind to their target RNAs with higher affinities than most other oligonucleotide chemistries. One measure of affinity is the melting temperature (Tm) for binding to its complementary sequence. For each LNA-modified nucleotide added to an ASO sequence, Tm can increase Tm by over 5°. For example, an LNA-modified oligonucleotide currently in clinical development, miravirsen (SPC3649), with 9 LNA-modified nucleotides has a Tm of approximately 80° demonstrating that LNA-modified oligonucleotides have sufficient affinities to bind to and inhibit RNA function. Affinities like these have translated into increase potency for the inhibition of target RNAs in nonclinical models and should yield therapeutic benefit more robust than those seen with earlier generations of oligonucleotide therapeutics. Other drug-like properties of antisense therapeutics have also been improved by including LNA-modified nucleotides. For example, the pharmacokinetic properties of antisense oligonucleotides support their use in clinical medicine. Antisense oligonucleotides are single stranded and bind to proteins in circulation and on cell surfaces. After parenteral administration, antisense oligonucleotide are bound first to plasma proteins, limiting glomerular filtration, and then later the antisense oligonucleotides appear bound to cell surface (proteins), allowing them to be internalized in cells. Delivery into cells and tissues is achieved without the need for formulations more complex than just aqueous solutions. LNA modifications increase the metabolic stability in both circulation and inside of cells resulting in stable tissue concentrations and prolonged activities. This long tissue residence and stability, allows for constant exposure in tumor cells with dosing as infrequent as weekly or fortnightly. Again, this represents an improvement over antisense drugs used in previous oncology trials. Using in vitro and in vivo laboratory models it has been possible to demonstrate that treatment with LNA-modified oligonucleotides produces reductions in target gene expression and ultimately tumor growth that are sequence-, concentration-, and duration-of-therapy-dependent. Taken together the properties of LNA-modified oligonucleotides make them excellent candidates for inhibiting key RNA targets and at this time there are multiple LNA-modified oligonucleotides in clinical trials in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY31-02. doi:10.1158/1538-7445.AM2011-SY31-02

Dual Masking of Specific Negative Splicing Regulatory Elements Resulted in Maximal Exon 7 Inclusion of SMN2 Gene

Editorial: In vitro mechanistic evaluation of nucleic acid polymers: A cautionary tale

The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2’-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3°C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50): 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50: 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.

Antisense oligodeoxynucleotides have been designed to inhibit the production of specific proteins. In models of hypertension, we have targeted the renin-angiotensin system at the level of synthesis (angiotensinogen) and the receptor (AT1 receptor). The design of antisense oligonucleotides requires choosing a site to inhibit mRNA processig or translation. The strategy we use is to make three oligonucleotides of antisense sequences, upstream and downstream from the AUG site and over the AUG site. The oligonucleotides are tested in a screening test. Antisense oligonucleotides to AT1-receptor mRNA and to angiotensinogen mRNA reduce blood pressure in spontaneously hypertensive rats when injected into the brain. They significantly reduce the concentration of the appropriate protein. The oligonucleotides are also effective when administered systemically. The decrease in blood pressure with antisense oligonucleotides delivered in blood or brain lasts 3 to 7 days. To prolong the action, direct injection of naked DNA and injection of DNA in liposome carriers have been tested. Viral vectors have been developed to deliver antisense DNA. The viral vectors available include retroviruses and adenovirus, but the adeno-associated virus (AAV) vector is the vector of choice for ultimate use in gene therapy. It offers safety because it is nonpathogenic, has longevity because it integrates into the genome, and has sufficient carrying capacity to carry up to 4.5 kb antisense or gene in a recombinant AAV. Using rAAV-antisense to AT1 mRNA, there is efficient transfection into cells and an inhibition of AT1 receptor number. In in vivo tests, rAAV-AS AT1-receptor when injected into the brains of SHR reduces blood pressure for more than 2 months. In young rats (3 weeks old), rAAV-AS AT1-receptor decreases blood pressure and slows the development of hypertension. While further experiments need to be done on dose-response relationships and on the cellular mechanisms of these effects, the results show the feasibility of AAV as a vector for antisense inhibition, which may ultimately be used in gene therapy for hypertension.