Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides (original) (raw)
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Molecular Therapy - Nucleic Acids
The introduction of sulfur into the phosphate linkage of chemically synthesized oligonucleotides creates the stereocenters on phosphorus atoms. Researchers have valued the nature of backbone stereochemistry and early on investigated drug properties for the individual stereocenters in dimers or short oligomers. Only very recently, it has become possible to synthesize fully stereodefined antisense oligonucleotides in good yield and purity. Non-bridging phosphorodithioate (PS 2) introduces second sulfur into the phosphorothioate linkage to remove the chirality of phosphorus atom. Here, we describe the application of symmetrical non-bridging PS 2 linkages in the context of stereodefined locked nucleic acids (LNAs) antisense oligonucleotides with the goal of reducing chiral complexity and, ultimately, resulting in single molecules. In addition, we propose a rather simple strategy to rapidly identify stereodefined gapmers, combining PS 2 and a preferred stereochemistry motif (RSSR), which supports RNase-H-mediated target knockdown. Pharmacological efficacy and metabolic stability are investigated systematically using ApoB as a target sequence, where in vivo data correlate well to what is observed in vitro.
Nucleic Acids Research, 2014
Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (T m), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Splinkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.
Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development
Journal of Clinical Medicine
Antisense oligonucleotides (ASOs) bind sequence specifically to the target RNA and modulate protein expression through several different mechanisms. The ASO field is an emerging area of drug development that targets the disease source at the RNA level and offers a promising alternative to therapies targeting downstream processes. To translate ASO-based therapies into a clinical success, it is crucial to overcome the challenges associated with off-target side effects and insufficient biological activity. In this regard, several chemical modifications and diverse delivery strategies have been explored. In this review, we systematically discuss the chemical modifications, mechanism of action, and optimized delivery strategies of several different classes of ASOs. Further, we highlight the recent advances made in development of ASO-based drugs with a focus on drugs that are approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for clinical applicatio...
Nucleic Acids Research, 2015
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 , 4bridged 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 ≈2fold increased efficacy, suggesting broad therapeutic applications.
Efficient synthesis of antisense phosphorothioate oligonucleotides using a universal solid support
Tetrahedron, 2006
It is demonstrated that solid support containing a novel universal linker could be efficiently used to synthesize both phosphorothioate oligodeoxyribonucleotides and second-generation 2 0 -O-methoxyethyloligoribonucleotides with high yield and quality as judged by ion-pair-liquid chromatography-electrospray mass spectroscopy, 31 P NMR and reversed phase HPLC. Analysis of oligonucleotides shows quality being superior to that produced with standard succinyl-linker solid supports, without contamination of materials resulting from linker or support backbone decomposition. q
Bioorganic & Medicinal Chemistry Letters, 1998
Antisense oligonucleotides are being evaluated in clinical trials as novel therapeutic agents. To further improve the properties of antisense oligonucleotides, we have designed mixed-backbone oligonucleotides (MBOs) that contain phosphorothioate segments at the 3 and 5 ends and have a modified oligodeoxynucleotide or oligoribonucleotide segment located in the central portion of the oligonucleotide. Some of these MBOs indicate improved properties compared with phosphorothioate oligodeoxynucleotides with respect to affinity to RNA, RNase H activation, and anti-HIV activity. In addition, more acceptable pharmacological, in vivo degradation and pharmacokinetic profiles were obtained with these MBOs.
Nucleic Acid Therapeutics
Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2¢-O-methyl (2¢-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2¢-methoxyethyl, 2¢ constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2¢-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5¢ end of the gap or in the 3¢-wing of a Gap 2¢-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2¢-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.
Mesyl Phosphoramidate Oligonucleotides: A New Promising Type of Antisense Agents
2023
This chapter provides an overview of mesyl phosphoramidate oligonucleotides (μ-oligonucleotides), which incorporate mesyl phosphoramidate groups (MsPA, or μ) instead of natural phosphate linkages up to the complete replacement at all internucleotidic positions. μ-Oligonucleotides can be efficiently obtained by β-cyanoethyl phosphoramidite chemistry via substituting Staudinger reaction of the intermediate phosphite triester with methanesulfonyl azide for conventional