Objective:In prior open-label studies, eteplirsen, a phosphorodiamidate morpholino oligomer, enabled dystrophin production in Duchenne muscular dystrophy (DMD) with genetic mutations amenable to skipping exon 51. The present study used a double-blind placebo-controlled protocol to test eteplirsen's ability to induce dystrophin production and improve distance walked on the 6-minute walk test (6MWT). Methods: DMD boys aged 7 to 13 years, with confirmed deletions correctable by skipping exon 51 and ability to walk 200 to 400 m on 6 MWT, were randomized to weekly intravenous infusions of 30 or 50 mg/kg/wk eteplirsen or placebo for 24 weeks (n 5 4/group). Placebo patients switched to 30 or 50 mg/kg eteplirsen (n 5 2/group) at week 25; treatment was open label thereafter. All patients had muscle biopsies at baseline and week 48. Efficacy included dystrophin-positive fibers and distance walked on the 6MWT. Results: At week 24, the 30 mg/kg eteplirsen patients were biopsied, and percentage of dystrophin-positive fibers was increased to 23% of normal; no increases were detected in placebo-treated patients (p 0.002). Even greater increases occurred at week 48 (52% and 43% in the 30 and 50 mg/kg cohorts, respectively), suggesting that dystrophin increases with longer treatment. Restoration of functional dystrophin was confirmed by detection of sarcoglycans and neuronal nitric oxide synthase at the sarcolemma. Ambulation-evaluable eteplirsen-treated patients experienced a 67.3 m benefit compared to placebo/delayed patients (p 0.001). Interpretation: Eteplirsen restored dystrophin in the 30 and 50 mg/kg/wk cohorts, and in subsequently treated, placebo-controlled subjects. Duration, more than dose, accounted for dystrophin production, also resulting in ambulation stability. No severe adverse events were encountered.
Systemically injected 2'-O-methoxyethyl (2'-O-MOE)-phosphorothioate and PNA-4K oligomers (peptide nucleic acid with four lysines linked at the C terminus) exhibited sequence-specific antisense activity in a number of mouse organs. Morpholino oligomers were less effective, whereas PNA oligomers with only one lysine (PNA-1K) were completely inactive. The latter result indicates that the four-lysine tail is essential for the antisense activity of PNA oligomers in vivo. These results were obtained in a transgenic mouse model designed as a positive readout test for activity, delivery, and distribution of antisense oligomers. In this model, the expressed gene (EGFP-654) encoding enhanced green fluorescence protein (EGFP) is interrupted by an aberrantly spliced mutated intron of the human beta-globin gene. Aberrant splicing of this intron prevented expression of EGFP-654 in all tissues, whereas in tissues and organs that took up a splice site-targeted antisense oligomer, correct splicing was restored and EGFP-654 expression upregulated. The sequence-specific ability of PNA-4K and the 2'-O-MOE oligomers to upregulate EGFP-654 provides strong evidence that systemically delivered, chemically modified oligonucleotides affect gene expression by sequence-specific true antisense activity, validating their application as potential therapeutics.
The antisense activity of oligomers with 2'-O-methyl (2'-O-Me) phosphorothioate, 2'-O-methoxyethyl (2'-O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2'-O-Me and 2'-O-MOE congeners. The effects of the PNA oligomers were observed to be dependent on the number of L-lysine (Lys) residues at the C-terminus. The experiments suggest that the PNA containing Lys was taken up by a mechanism similar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.
Polymeric nanogel vectors were developed for cellular gene and antisense delivery. Inverse microemulsion polymerization was utilized to synthesize biocompatible nanogels with controlled size, morphology, and composition. The chemical composition, size, polydispersity, stability, and swelling behavior of the nanogels were investigated by NMR, light scattering, transmission electron microscopy, and atomic force microscopy. The cell viability, uptake, and physical stability of nanogel-DNA complexes were evaluated under physiological conditions. Monodisperse nonionic and cationic nanogels were produced with controllable sizes ranging from 40 to 200 nm in diameter. The nanogels demonstrated extended stability in aqueous media and exhibited low toxicity in cell culture. Cationic nanogels formed monodisperse complexes with oligonucleotides and showed enhanced oligonucleotide uptake in cell culture. The nanogels synthesized in this study demonstrate potential utility as carriers of oligonucleotides and DNA for antisense and gene delivery.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.