Evaluation of 2’-Deoxy-2’-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON.

Using a State-of-the-Art Toolbox to Evaluate Molecular and Functional Readouts of Antisense Oligonucleotide-Induced Exon Skipping in mdx Mice

“…The concentration of M23D was determined using a hybridization/ligation enzyme-linked immunosorbent assay method based on an assay previously published [26] following adaptations previously described [27]. Briefly, tissues were homogenized in 100 mM Tris-HCl pH 8.5, 200 mM NaCl, 0.2% SDS, 5 mM ethylenediamine tetraaceticacid (all Sigma), and 2 mg/mL protK (ThermoFisher) using MagNA Lyser Green Beads (no.…”

Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B

“…We reasoned that shielding of the mutant site would prevent U1snRNP-binding and alleviate PAS suppression. Two types of antisense oligonucleotide (ASO) strategies previously demonstrated efficacy in other models: i) 2OMePS RNA oligos [32,38] with some side effects reported [39] and ii) an plasmid based system expressing an antisense oligo fused to U7 snRNA [35]. In essence, both antisense strategies lead to a rescue of F9 mRNA expression and an increase in coagulation activity, which may be able to revert the phenotype into a mild or subclinical hemophilia B. Interestingly, the RNA duplex formation and association with U7snRNP did not hamper translation in the cytosol, which suggests detachment of the complex at the nuclear pore or during the pioneer round of translation.…”

Nonclinical Exon Skipping Studies with 2′-O-Methyl Phosphorothioate Antisense Oligonucleotides in mdx and mdx-utrn−/− Mice Inspired by Clinical Trial Results

“…For measuring the concentration of the AON in plasma and tissue samples a hybridization/ligation enzyme-linked immunosorbent assay method based on an assay previously published was used [43] following adaptations previously described [44]. Briefly, equal tissue amounts were homogenized in 100 mM Tris-HCl, pH 8.5, 200 mM NaCl, 0.2% sodium dodecyl sulfate (SDS), 5 mM ethylenediaminetetraacetic acid and 2 mg/mL proteinase K using MagNa Lyzer green bead tubes in a MagNa Lyzer (Roche Diagnostics, the Netherlands).…”

“…Skeletal and cardiac muscles were homogenized in TRIsure buffer (GC Biotech, the Netherlands) using the MagNA Lyser and 1.4 mm Zirconium Beads Pre-Filled Tubes (OPS Diagnostics). This was followed by chloroform extraction as previously described [44]. For cDNA synthesis, 400 ng of RNA was used in a 20 μL reaction with random hexamer primers and transcriptor reverse transcriptase (Roche Diagnostics) for 45 min at 42°C.…”