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Stabilised alpha helical peptides: A novel platform for enhanced antisense oligonucleotide delivery

2016; Elsevier BV; Volume: 26; Linguagem: Inglês

10.1016/j.nmd.2016.06.154

1873-2364

Wouter Eilers, A. Gadd, Henry H. Foster, Alexander J. A. Cobb, Keith Foster,

Fuel Cells and Related Materials

Many novel therapeutic methods are being investigated for neuromuscular diseases and antisense oligonucleotide (AO) technologies to either target a toxic allele or induce RNA splicing suppression have particular promise. Current AO chemistries suffer from toxicity problems and/or insufficient cellular uptake when administered in vivo. To facilitate cell uptake, cell-penetrating peptides (CPPs) have been conjugated to PMOs. Unfortunately, the main property that enhances tissue transfection, i.e. a positively charged core, elicits membrane toxicity issues. We bring forward a platform technology that greatly increases the drug-likeness of peptides. Processes termed stapling and stitching of peptides in which two (stapled) or three (stitched) alpha, alpha-disubstituted, olefin-bearing amino acids are incorporated at defined positions during solid-phase peptide synthesis followed by all-hydrocarbon cross-links to stabilise the peptide into an alpha-helical structure have been developed. We demonstrate that stabilised alpha-helical peptide (SAP)-conjugation facilitates PMO uptake into mouse myotubes and subsequent skipping of dystrophin exon 23. In addition, we show that i/m-administered SAP-conjugated PMO is equally efficient in inducing exon skipping compared to unconjugated PMO in mdx mice (a pre-clinical model of DMD), thus alleviating potential concerns about steric hindrance. Finally, we show in preliminary experiments that a low dose systemic administration (intraperitoneal; 9 mg/kg) of SAP conjugated PMO (SAP-PMO) enhances dystrophin re-expression in diaphragm muscle. Importantly SAP-PMO administration leads to dystrophin re-expression in cardiac muscle and Purkinje cells of the cerebellum, tissues typically refractory to naked PMOs. We are currently conducting experiments to validate the efficacy of SAP-PMO in restoring systemic dystrophin expression, as well as establishing the mechanisms of cell entry of these novel conjugated PMOs.