Systematic evaluation of 2′-Fluoro modified chimeric antisense oligonucleotide-mediated exon skipping in vitro

Abstract Antisense oligonucleotide (AO)-mediated splice modulation has been established as a therapeutic approach for tackling genetic diseases. Recently, Exondys51, a drug that aims to correct splicing defects in the dystrophin gene was approved by the US Food and Drug Administration (FDA) for the treatment of Duchenne muscular dystrophy (DMD). However, Exondys51 has relied on phosphorodiamidate morpholino oligomer (PMO) chemistry which poses challenges in the cost of production and compatibility with conventional oligonucleotide synthesis procedures. One approach to overcome this problem is to construct the AO with alternative nucleic acid chemistries using solid-phase oligonucleotide synthesis via standard phosphoramidite chemistry. 2′-Fluoro (2′-F) is a potent RNA analogue that possesses high RNA binding affinity and resistance to nuclease degradation with good safety profile, and an approved drug Macugen containing 2′-F-modified pyrimidines was approved for the treatment of age-related macular degeneration (AMD). In the present study, we investigated the scope of 2′-F nucleotides to construct mixmer and gapmer exon skipping AOs with either 2′-O-methyl (2′-OMe) or locked nucleic acid (LNA) nucleotides on a phosphorothioate (PS) backbone, and evaluated their efficacy in inducing exon-skipping in mdx mouse myotubes in vitro. Our results showed that all AOs containing 2′-F nucleotides induced efficient exon-23 skipping, with LNA/2′-F chimeras achieving better efficiency than the AOs without LNA modification. In addition, LNA/2′-F chimeric AOs demonstrated higher exonuclease stability and lower cytotoxicity than the 2′-OMe/2′-F chimeras. Overall, our findings certainly expand the scope of constructing 2′-F modified AOs in splice modulation by incorporating 2′-OMe and LNA modifications

Antisense Oligonucleotides Targeting Angiogenic Factors as Potential Cancer Therapeutics

Cancer is one of the leading causes of death worldwide, and conventional cancer therapies such as surgery, chemotherapy, and radiotherapy do not address the underlying molecular pathologies, leading to inadequate treatment and tumor recurrence. Angiogenic factors, such as EGF, PDGF, bFGF, TGF-β, TGF-α, VEGF, endoglin, and angiopoietins, play important roles in regulating tumor development and metastasis, and they serve as potential targets for developing cancer therapeutics. Nucleic acid-based therapeutic strategies have received significant attention in the last two decades, and antisense oligonucleotide-mediated intervention is a prominent therapeutic approach for targeted manipulation of gene expression. Clinical benefits of antisense oligonucleotides have been recognized by the U.S. Food and Drug Administration, with full or conditional approval of Vitravene, Kynamro, Exondys51, and Spinraza. Herein we review the scope of antisense oligonucleotides that target angiogenic factors toward tackling solid cancers. Keywords: antisense oligonucleotides, nucleic acids, angiogenesis, antisense, modified oligonucleotides, cance

Synthesis and Biophysical Investigations of Oligonucleotides Containing Galactose-Modified DNA, LNA and 2'-Amino-LNA Monomers

Galactose-modified thymidine, LNA-T, and 2′-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2′-functionalized 2′-amino-LNA derivatives in particular, showed improved duplex thermal stability against DNA and RNA complements and increased ability to discriminate mismatches. In addition, the 2′-amino-LNA-T derivatives induced remarkable 3′-exonuclease resistance. These results were further investigated using molecular modeling studies.Foundation VILLUM; BIONEC (VKR022710); Ministerio de Economía y Competitividad (CTQ-2012-35360); German Research Foundation (DFG