A series of antisense oligonucleotides (ASOs) containing either 2′-O-methoxyethylribose (MOE) or locked nucleic acid (LNA) modifications were designed to investigate whether LNA antisense oligonucleotides (ASOs) have the potential to improve upon MOE based ASO therapeutics. Some, but not all, LNA containing oligonucleotides increased potency for reducing target mRNA in mouse liver up to 5-fold relative to the corresponding MOE containing ASOs. However, they also showed profound hepatotoxicity as measured by serum transaminases, organ weights and body weights. This toxicity was evident for multiple sequences targeting three different biological targets, as well as in mismatch control sequences having no known mRNA targets. Histopathological evaluation of tissues from LNA treated animals confirmed the hepatocellular involvement. Toxicity was observed as early as 4 days after a single administration. In contrast, the corresponding MOE ASOs showed no evidence for toxicity while maintaining the ability to reduce target mRNA. These studies suggest that while LNA ASOs have the potential to improve potency, they impose a significant risk of hepatotoxicity.
Abstract:The potency of second generation antisense oligonucleotides (ASOs) in animals was increased 3-to 5 -fold (ED 50 ≈ 2-5 mg/kg) without producing hepatotoxicity, by reducing ASO length (20-mer to 14-mer) and by employing novel nucleoside modifications that combine structural elements of 2′-O-methoxyethyl residues and locked nucleic acid. The ability to achieve this level of potency without any formulation agents is remarkable and likely to have a significant impact on the future design of ASOs as therapeutic agents.Antisense technology is a powerful method to modulate gene expression in animals and represents a novel therapeutic platform.1 The most advanced second generation antisense oligonucleotides (ASOs) are chimeric phosphorothioate 2,3 (PS) modified oligonucleotides, which have a central DNA region of 8-16 nucleotides, flanked on the 5′ and 3′ ends with five to two 2′-O-methoxyethyl (MOE) residues.4 This "gapmer" design supports RNase H mediated degradation of target mRNA due to the central DNA region. The flanking MOE residues increase hybridization to complementary mRNA and further stabilize the oligonucleotide toward enzymatic degradation. The PS backbone not only provides stabilization to nucleases but also confers a substantial pharmacokinetic benefit by increasing the binding to plasma proteins. This prevents rapid renal excretion of the ASO and facilitates binding to other acceptor sites which promote uptake to tissues. 5,6 There are currently multiple second generation ASOs in development for a variety of disease indications including hypercholesteremia, diabetes, and cancer, among others. One particular compound, mipomersen (ISIS 301012), targeting apolipoprotein B (ApoB), 7 reduced LDL cholesterol by 6-41% in normal volunteers at doses ranging from 50 to 400 (mg/kg)/ week in a phase I clinical trial. 8 Ongoing phase II clinical trials with mipomersen have further substantiated clinical efficacy and demonstrated an attractive safety profile for this drug. The improved performance of second generation ASOs in animals can be attributed in part to the higher affinity of MOE (B, Figure 1) residues (∆T m ≈ 1.5°C/incorporation), 10 which typically translates to increased binding affinity for the biological receptor (complementary mRNA). To probe if further increases in affinity could enhance the potency of gapmer ASOs, we replaced MOE residues in the wings with bicyclic nucleic acids such as 2′,4′-methylene bridged nucleic acid (BNA) 11 commonly called LNA (D, locked nucleic acids, Figure 1). 12 While this substitution resulted in improved potency of some ASOs in animals, it was accompanied by a significant increase in the risk for hepatotoxicity. 13 In contrast, the MOE modification employed in second generation ASOs is well tolerated in a variety of animal models and has also demonstrated an excellent safety profile in human clinical trials.14 Our previous study with LNA gapmers had indicated that the motifs that provided the greatest increase in potency of LNA containing ASOs were gapmer desi...
We show for the first time that it is possible to obtain LNA (Locked Nucleic Acid 1) like binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2’-oxygen atom in LNA with an exocyclic methylene group. Synthesis of the methylene-cLNA nucleoside was accomplished by an intramolecular cyclization reaction between a radical at the 2’-position and a propynyl group at C-4’ position. Only methylene-cLNA modified oligonucleotides showed similar thermal stability and mismatch discrimination properties for complementary nucleic acids as LNA. In contrast, the close structurally related methyl-cLNA analogs showed diminished hybridization properties. Analysis of crystal structures of cLNA modified self-complementary DNA decamer duplexes revealed that the methylene group participates in a tight interaction with a 2’-deoxyribose residue of the 5’-terminal G of a neighboring duplex, resulting in the formation of a CH…O type hydrogen bond. This indicates that the methylene group retains a negative polarization at the edge of the minor groove in the absence of a hydrophilic 2’-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA ASOs showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moeities in nucleic acid structure and emphasizes greater use of this bio-isostere to improve the properties of nucleic acids for therapeutic and diagnostic applications.
Antisense drug discovery technology is a powerful method to modulate gene expression in animals and represents a novel therapeutic platform.(1) We have previously demonstrated that replacing 2'O-methoxyethyl (MOE, 2) residues in second generation antisense oligonucleotides (ASOs) with LNA (3) nucleosides improves the potency of some ASOs in animals. However, this was accompanied with a significant increase in the risk for hepatotoxicity.(2) We hypothesized that replacing LNA with novel nucleoside monomers that combine the structural elements of MOE and LNA might mitigate the toxicity of LNA while maintaining potency. To this end we designed and prepared novel nucleoside analogs 4 (S-constrained MOE, S-cMOE) and 5 (R-constrained MOE, R-cMOE) where the ethyl chain of the 2'O-MOE moiety is constrained back to the 4' position of the furanose ring. As part of the SAR series, we also prepared nucleoside analogs 7 (S-constrained ethyl, S-cEt) and 8 (R-constrained Ethyl, R-cEt) where the methoxymethyl group in the cMOE nucleosides was replaced with a methyl substituent. A highly efficient synthesis of the nucleoside phosphoramidites with minimal chromatography purifications was developed starting from cheap commercially available starting materials. Biophysical evaluation revealed that the cMOE and cEt modifications hybridize complementary nucleic acids with the same affinity as LNA while greatly increasing nuclease stability. Biological evaluation of oligonucleotides containing the cMOE and cEt modification in animals indicated that all of them possessed superior potency as compared to second generation MOE ASOs and a greatly improved toxicity profile as compared to LNA.
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