Mesyl Phosphoramidate Oligonucleotides as Potential Splice-Switching Agents: Impact of Backbone Structure on Activity and Intracellular Localization

Abstract

A series of 2'-deoxy and novel 2'-O-methyl and 2'-O-(2-methoxyethyl) (2'-MOE) oligonucleotides with internucleotide methanesulfonyl (mesyl, μ) or 1butanesulfonyl (busyl, β) phosphoramidate groups have been synthesized for evaluation as potential splice-switching oligonucleotides. Evaluation of their spliceswitching activity in spinal muscular atrophy (SMA) patient derived fibroblasts revealed no significant difference in splice-switching efficacy between 2'-MOE mesyl oligonucleotide and the corresponding phosphorothioate (nusinersen). Yet, a survival study with model neonatal mice have shown the antisense 2'-MOE mesyl oligonucleotide to be inferior to nusinersen at the highest dose of 40 mg/kg. A reason for their lower activity in vivo as ascertained by cellular uptake study via fluorescent confocal microscopy in HEK293 cell line could possibly be ascribed to compromised endosomal release and/or nuclear uptake of the 2'-OMe or 2'-MOE μand β-oligonucleotides as compared to their phosphorothioate analog.

Synthesis of Oligonucleotides Carrying Inter-nucleotide N-(Benzoazole)-phosphoramide Moieties

“…This reaction between the esters of 3′,5′-dinucleoside phosphites and organic azides has been used previously to introduce N -sulfonylphosphoramide groups − and N -alkylphosphoramide groups , into the oligonucleotide. The phosphoryl guanidine and mesyl phosphoramidate oligonucleotides are particularly important as potential splice-switching agents …”

Phosphorothioate modification improves exon-skipping of antisense oligonucleotides based on sulfonyl phosphoramidates in mdx mouse myotubes

“…In summary, the present work and recently published article 48 show that various sulfonyl phosphoramidate oligonucleotides can be used in combination with other modifications to provide altered sugar-phosphate backbone and modulate the activity of oligonucleotides which can be applied for splice modulation.…”

“…It was also noted that the use of a different pentose sugar (2′-deoxy vs. 2′-OMe ribose vs. 2′-MOE-ribose) in combination with a different phosphate modification (PS vs. Ms- vs. Bs-) alters the level of endosomal release and cellular localisation, which is pivotal for exon-skipping processes occurring in the nucleus of the cell. 48 It was argued that lower activity of Ms- and Bs- 2′-OMe or 2′-MOE AOs in vivo could be explained by compromised endosomal release and/or nuclear uptake of these AOs in comparison with their PS-analogues. One should note that a combination of PS and phosphoramidate modifications in one AO was not evaluated in vitro and in vivo .…”

“…Very recently, Ms-and 1-butanesulfonyl (busyl, Bs-) phosphoramidates were incorporated into 2′-MOE AOs and tested in splice-switching activity in spinal muscular atrophy (SMA) in mice. 48 No significant differences in splice-switching efficacy between 2′-MOE Ms-or Bs-AOs and the corresponding PS AO, nusinersen, have been observed in mice and patientderived fibroblasts. It was also noted that the use of a different pentose sugar (2′-deoxy vs. 2′-OMe ribose vs. 2′-MOE-ribose) in combination with a different phosphate modification (PS vs. Ms-vs. Bs-) alters the level of endosomal release and cellular localisation, which is pivotal for exon-skipping processes occurring in the nucleus of the cell.…”

Single Shot vs. Cocktail: A Comparison of Mono- and Combinative Application of miRNA-Targeted Mesyl Oligonucleotides for Efficient Antitumor Therapy

“…To inhibit miRNA activity, we applied recently developed chemically modified antisense oligonucleotides, containing mesyl phosphoramidate (µ) modification of internucleotidic phosphates. These antisense µ-ONs were proven to exhibit high therapeutic potential in a number of studies [ 24 , 25 , 26 , 27 ]. Notably, our recent research showed the outstanding stability, superior RNase H-activating ability, long-lasting and specific downregulation of miRNA targets, and high biological performance of µ-oligonucleotides in tumor cells.…”