Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy

Abstract

The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD). In DMD patients, where major muscle loss has occurred due to a lack of dystrophin, the therapeutic restoration of dystrophin expression alone in older patients may not be sufficient to restore the functionality of the muscles. We recently demonstrated that phosphorodiamidate morpholino oligomers (PMOs) can be used to re-direct myostatin splicing and promote the expression of an out-of-frame transcript so reducing the amount of the synthesized myostatin protein. Furthermore, the systemic administration of the same PMO conjugated to an octaguanidine moiety (Vivo-PMO) led to a significant increase in the mass of soleus muscle of treated mice. Here, we have further optimized the use of Vivo-PMO in normal mice and also tested the efficacy of the same PMO conjugated to an arginine-rich cell-penetrating peptide (B-PMO). Similar experiments conducted in mdx dystrophic mice showed that B-PMO targeting myostatin is able to significantly increase the tibialis anterior (TA) muscle weight and when coadministered with a B-PMO targeting the dystrophin exon 23, it does not have a detrimental interaction. This study confirms that myostatin knockdown by exon skipping is a potential therapeutic strategy to counteract muscle wasting conditions and dual myostatin and dystrophin skipping has potential as a therapy for DMD.

Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice

“…We have initially published that AO-induced out-of-frame skipping of myostatin stimulated hypertrophy in treated tibialis anterior muscles of wild-type mice 15 . We and others have further investigated dual skipping of dystrophin and myostatin in mouse and human dystrophic cells 16 and in a mdx mouse model of DMD 10 , confirming the efficacy of the combined antisense therapy with no interference between the two AOs. However, BPMO-induced exon skipping levels of myostatin in treated mice were 4-5 times lower than of dystrophin although both BPMOs were used at the same dose, resulting in no significant difference in the therapeutic effects between single and dual treatments.…”

“…In order to confirm our previous findings 10 and to further enhance the therapeutic benefits of antisense oligonucleotide (AO)-induced dual exon skipping of dystrophin and myostatin pre-mRNAs, we investigated here a combined therapy in p0 mdx mice through regular intraperitoneal injection. Mdx neonates were injected with 10 mg/kg of BPMO-DMD ( n = 7) or a mixture of BPMO-DMD and BPMO-MSTN, 10 mg/kg each BPMO ( n = 6).…”

“…Following our previous encouraging findings 10,15 , we investigated the effect of antisense-mediated dual exon skipping of dystrophin and myostatin pre-mRNAs in neonatal mdx mice. After three triweekly injections, we detected exon skipping of dystrophin (16-78%) and myostatin (5-29%) in all examined BPMO-treated muscles despite a high variability in the skipping level between muscle types and between subjects receiving the same treatment.…”

“…PMOs conjugated with an arginine-rich B peptide (BPMOs). Pre-clinical tests in DMD mouse models have demonstrated therapeutic effects of BPMOs were far more efficient than of unconjugated PMOs when used at the same dose 10 , and interestingly with dystrophin expression in body-wide skeletal plus cardiac muscles 11 .…”

Targeted Excision of VCP R155H Mutation by Cre-LoxPTechnology as a Promising Therapeutic Strategy for Valosin-Containing Protein Disease

“…40 Recently, exon skipping as a therapeutic platform has demonstrated successful treatment in DMD mice and patients. [41][42][43] Here, we describe our novel tamoxifen-inducible Cre-ER TM -VCP R155H/ + mouse line as a powerful tool and show that targeted excision of the R155H mutation in exons 4 and 5 ameliorates the phenotype typically observed in patients with VCP-associated disease. The rationale for deletion of exons 4 and 5 was to disrupt the mutant VCP open reading frame, thereby leaving the normal allele intact, providing proof of principle that removal of the mutated gene could result in the improvement of muscle and brain pathologies.…”