Design of antisense oligonucleotides stabilized by locked nucleic acids

The design of antisense oligonucleotides containing locked nucleic acids (LNA) was optimized and compared to intensively studied DNA oligonucleotides, phosphorothioates and 2′-O-methyl gapmers. In contradiction to the literature, a stretch of seven or eight DNA monomers in the center of a chimeric DNA/LNA oligonucleotide is necessary for full activation of RNase H to cleave the target RNA. For 2′-O-methyl gapmers a stretch of six DNA monomers is sufficient to recruit RNase H. Compared to the 18mer DNA the oligonucleotides containing LNA have an increased melting temperature of 1.5–4°C per LNA depending on the positions of the modified residues. 2′-O-methyl nucleotides increase the T_m by only <1°C per modification and the T_m of the phosphorothioate is reduced. The efficiency of an oligonucleotide in supporting RNase H cleavage correlates with its affinity for the target RNA, i.e. LNA > 2′-O-methyl > DNA > phosphorothioate. Three LNAs at each end of the oligonucleotide are sufficient to stabilize the oligonucleotide in human serum 10-fold compared to an unmodified oligodeoxynucleotide (from t_1/2 = ~1.5 h to t_1/2 = ~15 h). These chimeric LNA/DNA oligonucleotides are more stable than isosequential phosphorothioates and 2′-O-methyl gapmers, which have half-lives of 10 and 12 h, respectively.     

RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

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.     

Inhibition of MDR1 gene expression by chimeric HNA antisense oligonucleotides

Hexitol nucleic acids (HNAs) are nuclease resistant and provide strong hybridization to RNA. However, there is relatively little information on the biological properties of HNA antisense oligonucleotides. In this study, we compared the antisense effects of a chimeric HNA ‘gapmer’ oligonucleotide comprising a phosphorothioate central sequence flanked by 5′ and 3′ HNA sequences to conventional phosphorothioate oligonucleotides and to a 2′-O-methoxyethyl (2′-O-ME) phosphorothioate ‘gapmer’. The antisense oligomers each targeted a sequence bracketing the start codon of the message of MDR1, a gene involved in multi-drug resistance in cancer cells. Antisense and control oligonucleotides were delivered to MDR1-expressing cells using transfection with the cationic lipid Lipofectamine 2000. The anti-MDR1 HNA gapmer was substantially more potent than a phosphorothioate oligonucleotide of the same sequence in reducing expression of P-glycoprotein, the MDR1 gene product. HNA and 2′-O-ME gapmers displayed similar potency, but a pure HNA antisense oligonucleotide (lacking the phosphorothioate ‘gap’) was ineffective, indicating that RNase H activity was likely required. Treatment with anti-MDR1 HNA gapmer resulted in increased cellular accumulation of the drug surrogate Rhodamine 123 that correlated well with the reduced cell surface expression of P-glycoprotein. Thus, HNA gapmers may provide a valuable additional tool for antisense-based investigations and therapeutic approaches.     

Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design

Use of antisense oligonucleotides is a versatile strategy for achieving control of gene expression. Unfortunately, the interpretation of antisense-induced phenotypes is sometimes difficult, and chemical modifications that improve the potency and specificity of antisense action would be useful. The introduction of locked nucleic acid (LNA) bases into oligonucleotides confers exceptional improvement in binding affinity, up to 10°C per substitution, making LNAs an exciting option for the optimization of antisense efficacy. Here we examine the rules governing antisense gene inhibition within cells by oligonucleotides that contain LNA bases. LNA- containing oligomers were transfected into cells using cationic lipid and accumulated in the nucleus. We tested antisense gene inhibition by LNAs and LNA–DNA chimeras complementary to the 5′-untranslated region, the region surrounding the start codon and the coding region of mRNA, and identified effective antisense agents targeted to each of these locations. Our data suggest that LNA bases can be used to develop antisense oligonucleotides and that their use is a versatile approach for efficiently inhibiting gene expression inside cells.     

2'-O-[2-[(N,N-dimethylamino)oxy]ethyl]-modified oligonucleotides inhibit expression of mRNA in vitro and in vivo

Synthesis and antisense activity of oligonucleotides modified with 2′-O-[2-[(N,N-dimethylamino)oxy] ethyl] (2′-O-DMAOE) are described. The 2′-O-DMAOE-modified oligonucleotides showed superior metabolic stability in mice. The phosphorothioate oligonucleotide ‘gapmers’, with 2′-O-DMAOE- modified nucleoside residues at the ends and 2′-deoxy nucleosides residues in the central region, showed dose-dependent inhibition of mRNA expression in cell culture for two targets. ‘Gapmer’ oligonucleotides have one or two 2′-O-modified regions and a 2′-deoxyoligonucleotide phosphorothioate region that allows RNase H digestion of target mRNA. To determine the in vivo potency and efficacy, BalbC mice were treated with 2′-O-DMAOE gapmers and a dose-dependent reduction in the targeted C-raf mRNA expression was observed. Oligonucleotides with 2′-O-DMAOE modifications throughout the sequences reduced the intercellular adhesion molecule-1 (ICAM-1) protein expression very efficiently in HUVEC cells with an IC₅₀ of 1.8 nM. The inhibition of ICAM-1 protein expression by these uniformly modified 2′-O-DMAOE oligonucleotides may be due to selective interference with the formation of the translational initiation complex. These results demonstrate that 2′-O-DMAOE- modified oligonucleotides are useful for antisense-based therapeutics when either RNase H-dependent or RNase H-independent target reduction mechanisms are employed.     

Insights into structure, dynamics and hydration of locked nucleic acid (LNA) strand-based duplexes from molecular dynamics simulations

Locked nucleic acid (LNA) is a chemically modified nucleic acid with its sugar ring locked in an RNA-like (C3′-endo) conformation. LNAs show extraordinary thermal stabilities when hybridized with DNA, RNA or LNA itself. We performed molecular dynamics simulations on five isosequential duplexes (LNA–DNA, LNA–LNA, LNA–RNA, RNA–DNA and RNA–RNA) in order to characterize their structure, dynamics and hydration. Structurally, the LNA–DNA and LNA–RNA duplexes are found to be similar to regular RNA–DNA and RNA–RNA duplexes, whereas the LNA–LNA duplex is found to have its helix partly unwound and does not resemble RNA–RNA duplex in a number of properties. Duplexes with an LNA strand have on average longer interstrand phosphate distances compared to RNA–DNA and RNA–RNA duplexes. Furthermore, intrastrand phosphate distances in LNA strands are found to be shorter than in DNA and slightly shorter than in RNA. In case of induced sugar puckering, LNA is found to tune the sugar puckers in partner DNA strand toward C3′-endo conformations more efficiently than RNA. The LNA–LNA duplex has lesser backbone flexibility compared to the RNA–RNA duplex. Finally, LNA is less hydrated compared to DNA or RNA but is found to have a well-organized water structure.     

Inhibition of telomerase by 2'-O-(2-methoxyethyl) RNA oligomers: effect of length, phosphorothioate substitution and time inside cells

2′-O-(2-methoxyethyl) (2′-MOE) RNA possesses favorable pharmocokinetic properties that make it a promising option for the design of oligonucleotide drugs. Telomerase is a ribonucleoprotein that is up-regulated in many types of cancer, but its potential as a target for chemotherapy awaits the development of potent and selective inhibitors. Here we report inhibition of human telomerase by 2′-MOE RNA oligomers that are complementary to the RNA template region. Fully complementary oligomers inhibited telomerase in a cell extract with IC₅₀ values of 5–10 nM at 37°C. IC₅₀ values for mismatch-containing oligomers varied with length and phosphorothioate substitution. After introduction into DU 145 prostate cancer cells inhibition of telomerase activity persisted for up to 7 days, equivalent to six population doublings. Inside cells discrimination between complementary and mismatch-containing oligomers increased over time. Our results reveal two oligomers as especially promising candidates for initiation of in vivo preclinical trials and emphasize that conclusions regarding oligonucleotide efficacy and specificity in cell extracts do not necessarily offer accurate predictions of activity inside cells.     

Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

Oligonucleotides composed of 2′-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5′-disulphide-linked conjugates of 3′-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48–58), Penetratin and R₉F₂ was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48–58), Penetratin and R₉ conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R₉F₂ and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide.     

Intracellular inhibition of hepatitis C virus (HCV) internal ribosomal entry site (IRES)-dependent translation by peptide nucleic acids (PNAs) and locked nucleic acids (LNAs)

Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B hepatitis. Current therapies are not effective in all patients and can result in the generation of resistant mutants, leading to a need for new therapeutic options. HCV has an RNA genome that contains a well-defined and highly conserved secondary structure within the 5′-untranslated region. This structure is known as the internal ribosomal entry site (IRES) and is necessary for translation and viral replication. Here, we test the hypothesis that antisense peptide nucleic acid (PNA) and locked nucleic acid (LNA) oligomers can bind key IRES sequences and block translation. We used lipid-mediated transfections to introduce PNAs and LNAs into cells. Our data suggest that PNAs and LNAs can invade critical sequences within the HCV IRES and inhibit translation. Seventeen base PNA or LNA oligomers targeting different regions of the HCV IRES demonstrated a sequence-specific dose–response inhibition of translation with EC₅₀ values of 50–150 nM. Inhibition was also achieved by PNAs ranging in length from 15 to 21 bases. IRES-directed inhibition of gene expression widens the range of mechanisms for antisense inhibition by PNAs and LNAs and may provide further therapeutic lead compounds for the treatment of HCV.     

Selective inhibitory DNA aptamers of the human RNase H1

Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA–DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K_d values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5′ and 3′ tails that form a stem of six base pairs. Base pairing between the 5′ and 3′ tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC₅₀ ranging from 50 to 100 nM.     

Hydration of short DNA, RNA and 2'-OMe oligonucleotides determined by osmotic stressing

Studies on hydration are important for better understanding of structure and function of nucleic acids. We compared the hydration of self-complementary DNA, RNA and 2′-O-methyl (2′-OMe) oligonucleotides GCGAAUUCGC, (UA)₆ and (CG)₃ using the osmotic stressing method. The number of water molecules released upon melting of oligonucleotide duplexes, Δn_W, was calculated from the dependence of melting temperature on water activity and the enthalpy, both measured with UV thermal melting experiments. The water activity was changed by addition of ethylene glycol, glycerol and acetamide as small organic co-solutes. The Δn_W was 3–4 for RNA duplexes and 2–3 for DNA and 2′-OMe duplexes. Thus, the RNA duplexes were hydrated more than the DNA and the 2′-OMe oligonucleotide duplexes by approximately one to two water molecules depending on the sequence. Consistent with previous studies, GC base pairs were hydrated more than AU pairs in RNA, whereas in DNA and 2′-OMe oligonucleotides the difference in hydration between these two base pairs was relatively small. Our data suggest that the better hydration of RNA contributes to the increased enthalpic stability of RNA duplexes compared with DNA duplexes.     

Thermodynamic and Kinetic Characterization of Duplex Formation between 2′-O, 4′-C-Methylene-modified Oligoribonucleotides,DNA and RNA

2′-O,4′-C-methylene-linked ribonucleotide derivatives, named LNA (locked nucleic acid) and BNA (bridged nucleic acid) are nucleic acid analogoues that have shown high-affinity recognition of DNA and RNA, and the employment of LNA oligomers for antisense activity, gene regulation and nucleic acid diagnostics seems promising. Here we show kinetic and thermodynamic results on the interaction of a series of 10 bases long LNA–DNA mixmers, gabmers as well as full length LNA’s with the complementary DNA, RNA and LNA oligonucleotides in the presence and absence of 10 mM Mg²⁺- ions. Our results show no significant differences in the reaction thermodynamics and kinetics between the LNA species, only a tendency to stronger duplex formation with the gabmer and mixmer. Introduction of a few LNA’s thus may be a better strategy, than using full length LNA’s to obtain an oligonucleotide that markedly increases the strength of duplexes formed with the complementary DNA and RNA.     

Enhancing antisense efficacy with multimers and multi-targeting oligonucleotides (MTOs) using cleavable linkers

The in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8–15 nucleotides and by the incorporation of high affinity RNA binders such as 2′, 4′-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2′,4′-constrained ethyl [cET]). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4–5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications.     

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1–2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.     

The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides

The biological activity of siRNA seems to be influenced by local characteristics of the target RNA, including local RNA folding. Here, we investigated quantitatively the relationship between local target accessibility and the extent of inhibition of the target gene by siRNA. Target accessibility was assessed by a computational approach that had been shown earlier to be consistent with experimental probing of target RNA. Two sites of ICAM-1 mRNA predicted to serve as accessible motifs and one site predicted to adopt an inaccessible structure were chosen to test siRNA constructs for suppression of ICAM-1 gene expression in ECV304 cells. The local target-dependent effectiveness of siRNA was compared with antisense oligonucleotides (asON). The concentration dependency of siRNA-mediated suppression indicates a >1000-fold difference between active siRNAs (IC₅₀ ≈ 0.2–0.5 nM) versus an inactive siRNA (IC₅₀ ≥ 1 µM) which is consistent with the activity pattern of asON when relating target suppression to predicted local target accessibility. The extremely high activity of the siRNA si2B (IC₅₀ = 0.24 nM) indicates that not all siRNAs shown to be active at the usual concentrations of >10–100 nM belong to this highly active species. The observations described here suggest an option to assess target accessibility for siRNA and, thus, support the design of active siRNA constructs. This approach can be automated, work at high throughput and is open to include additional parameters relevant to the biological activity of siRNA.     

Antisense oligonucleotides targeted to the domain IIId of the hepatitis C virus IRES compete with 40S ribosomal subunit binding and prevent in vitro translation

Initiation of protein synthesis on the hepatitis C virus (HCV) mRNA involves a structured element corresponding to the 5′ untranslated region and constituting an internal ribosome entry site (IRES). The domain IIId of the HCV IRES, an imperfect RNA hairpin extending from nucleotides 253 to 279 of the viral mRNA, has been shown to be essential for translation and for the binding of the 40S ribosomal subunit. We investigated the properties of a series of antisense 2′-O-methyloligoribonucleotides targeted to various portions of the domain IIId. Several oligomers, 14–17 nt in length, selectively inhibited in vitro translation of a bicistronic RNA construct in rabbit reticulocyte lysate with IC₅₀s <10 nM. The effect was restricted to the second cistron (the Renilla luciferase) located downstream of the HCV IRES; no effect was observed on the expression of the first cistron (the firefly luciferase) which was translated in a cap-dependent manner. Moreover, antisense 2′-O-methyloligoribonucleotides specifically competed with the 40S ribosomal subunit for binding to the IRES RNA in a filter- retention assay. The antisense efficiency of the oligonucleotides was nicely correlated to their affinity for the IIId subdomain and to their ability to displace 40S ribosomal subunit, making this process a likely explanation for in vitro inhibition of HCV-IRES-dependent translation.     

Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5'-protected thiol function and a 3'-amino group

A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphorothioate oligonucleotides substituted with a protected thiol function at their 5′-ends and an amino group at their 3′-ends in good yield (up to 72 OD units/µmol for a 19mer phosphorothioate). Syntheses of 3′-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphoramidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2′-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This procedure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3′-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5′-terminal S-acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3′-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligonucleotide; the 5′-dithiopyridyl group was then quantitatively reduced to give a free thiol group which was then substituted by reaction with an Nα-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide–oligonucleotide conjugate.     

The high binding affinity of phosphorothioate-modified oligomers for Ff gene 5 protein is moderated by the addition of C-5 propyne or 2′-O-methyl modifications

One of the problems that hamper the use of antisense DNAs as effective drugs is the non-specific binding of chemically-modified oligonucleotides to cellular proteins. We previously showed that the affinity of a model ssDNA-binding protein, the Ff gene 5 protein (g5p), was >300-fold higher for phosphorothioate-modified DNA (S-DNA) than for unmodified dA₃₆, consistent with the propensity of S-DNA to bind indiscriminately to proteins. The current work shows that g5p binding is also sensitive to sugar and pyrimidine modifications used in antisense oligomers. Binding affinities of g5p for 10 36mer oligomers were quantitated using solution circular dichroism measurements. The oligomers contained C-5-propyne (prC), 2′-O-methyl (2′-O-Me) or 2′-OH (RNA) groups, alone or combined with the phosphorothioate modification. In agreement with reported increases in antisense activity, the addition of prC or 2′-O-Me modifications substantially reduced the affinity of oligomers for g5p by ~2-fold compared with the same DNA oligomer sequences containing only phosphorothioate linkages. That is, such modifications moderated the propensity of the phosphorothioate group to bind tightly to the g5p. The Ff g5p could be a useful model protein for assessing non-specific binding effects of antisense oligomer modifications.