Synthesis of a Morpholino Nucleic Acid (MNA)-Uridine Phosphoramidite, and Exon Skipping Using MNA/2′-O-Methyl Mixmer Antisense Oligonucleotide

Abstract

Abstract:In this study, we synthesised a morpholino nucleoside-uridine (MNA-U) phosphoramidite and evaluated the potential of a MNA-modified antisense oligonucleotide (AO) sequences to induce exon 23 skipping in mdx mouse myotubes in vitro towards extending the applicability of morpholino chemistry with other nucleotide monomers. We designed, synthesised, and compared exon skipping efficiencies of 20 mer MNA-modified 2 -O-methyl RNA mixmer AO on a phosphorothioate backbone (MNA/2 -OMePS) to the corresponding fully modified 2 -O-methyl RNA AO (2 -OMePS) as a control. Our results showed that the MNA/2 -OMePS efficiently induced exon 23 skipping. As expected, the 2 -OMePS AO control yielded efficient exon 23 skipping. Under the applied conditions, both the AOs showed minor products corresponding to exon 22/23 dual exon skipping in low yield. As these are very preliminary data, more detailed studies are necessary; however, based on the preliminary results, MNA nucleotides might be useful in constructing antisense oligonucleotides.

Antisense oligonucleotide modified with serinol nucleic acid (SNA) induces exon skipping in mdx myotubes

“…4,5 In addition, PNA also shows very high stability against nucleases. 5,6 Recently, the potential of different chemically-modied antisense oligonucleotides (AOs) including twisted intercalating nucleic acids (TINA), 7 anhydrohexitol nucleic acid (HNA), 8 cyclohexenyl nucleic acid (CeNA), 8 altritol nucleic acid (ANA), 8 morpholino nucleic acid (MNA) 9 and also PNA 10,11 has been explored in several studies for exon-skipping in Duchenne muscular dystrophy (DMD), a muscle wasting fatal genetic disease mainly affecting boys caused by the mutations in the dystrophin gene. 12,13 However, based on previous reports, the cell delivery of PNA AOs using normal lipid-based transfection reagents is difficult, mainly due to the charge issue.…”

2′-O-Methyl RNA/Ethylene-Bridged Nucleic Acid Chimera Antisense Oligonucleotides to Induce Dystrophin Exon 45 Skipping

“…Subsequently, in order to develop successful AO-mediated exon skipping therapy, various kinds of modified AOs have been chemically synthesized, with some of the most prominent being phosphorodiamidate morpholino oligonucleotides (PMOs), 2′- O -methyl (2′-OMe) AOs, locked nucleic acid (LNA), peptide nucleic acid (PNA), and tricycle-DNA (tcDNA) [ 10 , 11 ]. Furthermore, chimera AOs such as anhydrohexitol nucleic acid (HNA)/2′-OMe phosphorothioate (2′OMePS), cyclohexenyl nucleic acid (CeNA)/2′OMePS, altritol nucleic acid (ANA)/2′OMePS, and morpholino nucleic acid (MNA)/2′OMePS have been investigated [ 12 , 13 ]. Among these AOs, two AOs comprising different monomers—PMOs [ 14 ] and 2′-OMePS [ 15 ]—were developed to induce skipping of dystrophin exon 51 and have undergone clinical trials.…”

Alpha-l-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce Efficient Splice Modulation In Vitro

“…Another drug candidate entered in Phase 3 trials, Drisapersen (2′-OMePS chemistry), was rejected by the FDA due to the failure to meet the primary and secondary endpoints and also due to the increased renal and hepatotoxicity [14], which reinforces the requirement for alternative approaches to improve the efficacy of the 2′-OMePS chemistry. Towards this goal, various chemically-modified nucleic acid analogues have been explored previously [16][17][18][19][20][21][22][23]. In this study, we explored the scope of α-L-LNA analogues to induce exon-skipping.…”