Efficacy of systemic morpholino exon‐skipping in duchenne dystrophy dogs

Abstract

Objective Duchenne muscular dystrophy (DMD) is caused by the inability to produce dystrophin protein at the myofiber membrane. A method to rescue dystrophin production by antisense oligonucleotides, termed `exon-skipping', has been reported for the mdx mouse and in four DMD patients by local intramuscular injection. We sought to test efficacy and toxicity of intravenous oligonucleotide (morpholino) induced exon skipping in the DMD dog model. Methods We tested a series of antisense drugs singly and as cocktails, both in primary cell culture, and two in vivo delivery methods (intramuscular injection, and systemic intravenous injection). The efficiency and efficacy of multi-exon skipping (exons 6-9) was tested at the mRNA, protein, histological, and clinical levels. Results Weekly or biweekly systemic intravenous injections with a three morpholino cocktail over the course of 5–22 weeks induced therapeutic levels of dystrophin expression throughout the body, with an average of about 26% normal levels. This was accompanied by reduced inflammatory signals examined by MRI and histology, improved or stabilized timed running tests and clinical symptoms. Blood tests indicated no evidence of toxicity. Interpretation This is the first report of widespread rescue of dystrophin expression to therapeutic levels in the dog model of DMD. This study also provides a proof of concept for systemic multi-exon skipping therapy. Use of cocktails of morpholinos, as shown here, allows broader application of this approach to a greater proportion of DMD patients (90%), and also offers the prospect of selecting deletions that optimize the functionality of the dystrophin protein.

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

“…10 Similar dose effect of morpholino for dystrophin exon skipping has also been observed in the dystrophic dogs. 11 Thus, regular treatment of DMD with 80 mg kg -1 or below could rescue the dystrophic skeletal muscles, but is most likely insufficient for rescue of cardiac dysfunctions. This study shows that at the dose of 0.3 g kg -1 , morpholinoE23 can achieve approximately 5% dystrophin induction in heart muscles.…”

“…This can lead to the production of truncated but functional dystrophin, and can reverse a DMD to a milder Becker muscular dystrophy or near normal phenotypes. [8][9][10][11] The therapeutic potential of antisense therapy for DMD was initially showed in the dystrophic mdx mouse that harbors a nonsense point mutation in the exon 23, leading to general absence of dystrophin in the muscles. Specifically designed 2 0 O methyl phosphorothioate AONs delivered by intramuscular injections were able to skip the exon 23 effectively in tibialis anterior (TA) muscles and produce functional amount of proteins.…”

“…10 Systemic effect of morpholino oligomers for exon skipping has now been shown in dystrophic dog. 11 The dog harbors a splice site mutation in intron 6, leading to exclusion of exon 7 from the mRNA transcript. Treating the dogs weekly or biweekly with a cocktail of three morpholino oligomers (120-200 mg kg -1 ) systemically resulted in further removal of exon 6 and 8 with extensive dystrophin expression and significant functional stabilization by several criteria.…”

“…Treating the dogs weekly or biweekly with a cocktail of three morpholino oligomers (120-200 mg kg -1 ) systemically resulted in further removal of exon 6 and 8 with extensive dystrophin expression and significant functional stabilization by several criteria. 11 Furthermore, effective restoration of reading frame and production of dystrophin have now been achieved in Phase I clinical trials targeting human dystrophin exon 51 in muscles of DMD patients by local injections with both 2 0 O methyl phosphorothioate AONs and morpholino oligomers (personal communication). 12,13 DMD affects body-wide muscles including the cardiac muscle.…”

Épissothérapie et thalassémies

“…C'est pourquoi on a en parallèle tenté d'obtenir une production permanente in situ des AON correcteurs grâce à des constructions camouflées dans une séquence naturelle d'ARN nucléolaire ou snARN, en particulier la séquence U7, et incorporées dans un vecteur viral [21]. Cette stratégie a déjà fait la preuve expérimentale de son efficacité prolongée pour le saut d'exon thérapeutique dans un modèle murin de dystrophinopathie [22,[23][24][25][26]. Mais ici, …”