Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

Abstract

Antisense oligonucleotide-mediated exon skipping is able to correct out-of-frame mutations in Duchenne muscular dystrophy and restore truncated yet functional dystrophins. However, its application is limited by low potency and inefficiency in systemic delivery, especially failure to restore dystrophin in heart. Here, we conjugate a phosphorodiamidate morpholino oligomer with a designed cellpenetrating peptide (PPMO) targeting a mutated dystrophin exon. Systemic delivery of the novel PPMO restores dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. This leads to increase in muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo. Muscle pathology and function continue to improve during the 12-week course of biweekly treatment, with significant reduction in levels of serum creatine kinase. The high degree of potency of the oligomer in targeting all muscles and the lack of detectable toxicity and immune response support the feasibility of testing the novel oligomer in treating Duchenne muscular dystrophy patients.M utations in the dystrophin gene underlie two forms of muscular dystrophy: Duchenne and Becker muscular dystrophy (DMD and BMD). DMD is caused mainly by nonsense and frame-shift mutations with little or no production of functional dystrophin protein, leading to disease onset in early childhood with lethal consequences. BMD is caused by mutations that typically create shortened but in-frame transcripts with production of partially functional dystrophin, leading to variable and often overt symptoms (1-3). Most DMD mutations occur within the rod domain, which spans more than half the length of the protein, but seems to have limited functional importance (4, 5). Antisense therapy uses specific oligomers to remove the mutated or additional exon(s) that disrupt the reading frame, thus restoring the expression of shortened forms of dystrophin protein retaining critical functions (6-11).We previously demonstrated that i.m. delivery of a specific 2Ј-O-methyl phosphorothioate antisense oligonucleotide (2ЈOMeAON) was able to skip targeted dystrophin exon 23 in mdx mouse, a model of DMD (9). This mouse carries a nonsense point mutation within exon 23 and lacks dystrophin expression (except in a few rare revertant fibers) in all muscles, including the heart (12, 13). Skipping the mutated exon 23 restored both the reading frame and dystrophin expression, with functional improvement of the treated muscles (14) [supporting information (SI) Fig. S1a]. Recently we showed that a phosphorodiamidate morpholino oligomer (PMO), E23ϩ7-18 targeting the junction of exon 23 and intron 23 of mouse dystrophin (referred to as PMOE23 hereafter), was able to induce up to functional levels of dystrophin expression in some skeletal muscles by regular i.v. injections in mdx mice (15). However, dystrophin expression induced by both 2ЈOMeAON and PMO required high doses and was highly variable between muscles and myofibers in terms of observed efficacy. Of greater conc...

Dose-dependent restoration of dystrophin expression in cardiac muscle of dystrophic mice by systemically delivered morpholino

“…Specifically, the potential of immune response to the delivery enabling peptides needs to be assessed with long-term administration in animal models and ultimately by clinic trials. 21 More recently, we reported that a non-peptide cationic polymer, Octa-guanidine, also significantly improves morpholino-mediated exon skipping and dystrophin expression in both cardiac and skeletal muscles, resulting in amelioration in pathology of dystrophic mdx mice. 22 However, considerable increase in toxicity is expected when the cationic polymers are delivered systemically, although no clear toxicity is observed at the reported doses for a relatively short time period.…”

“…21,22 Systemic delivery of a morpholino conjugated with the peptide of (RXRRBR)2XB sequence Dose-dependent restoration of dystrophin expression B Wu et al (R:arginine, X:6-aminohexanoic acid, and B:alanine) is able to restore dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. The treatment also increases muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo.…”

“…Nevertheless, the results show that the charge-neutral morphoplino can enter the cardiac myocytes and induce effective and specific exon skipping, suggesting that degeneration process is not a prerequisite for effective rescue of dystrophin by unmodified morpholino. 21 Determination of effective dosage is a prerequisite for clinic applications of any potential drug. A clinic trial with morpholino for dystrophin exon 51 skipping is being conducted in UK for the treatment of DMD patients (personal communication).…”

“…21,22 The protein concentration was quantified by Figure 6 Examination of pathology and serum testing after morpholinoE23 treatment. (a) Histology of TA, diaphragm and kidney (hematoxylin and eosin staining) from the normal C57BL6 mice (C57), untreated mdx mice (Untreated), 3 g kg -1 morpholinoE23-treated mdx mice (3 g kg -1 treated).…”

In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy

“…This has prompted the search for improved splice correcting AO chemistries [e.g. neutrally charged peptide nucleic acid (PNA) AOs] [8] and the investigation of novel AO-peptide conjugate chemistries [8,9,[13][14][15][16][17].…”