Steve Wilton AO FAHMS BSc PhD

Duchenne muscular dystrophy (DMD) is an X-linked, relentlessly progressive muscle wasting disorder, caused by mutations in the DMD gene that compromise function of dystrophin, a sub sarcolemmal protein. DMD has a predictable course and limited treatment options, with the majority of cases being caused by frame-shifting deletions of one or more of the 79 exons of DMD. Deletions that do not disrupt the dystrophin open reading frame generally cause the milder allelic disorder, Becker muscular dystrophy (BMD). Antisense oligomer (AO)-mediated splicing manipulation can remove specific exons during transcript processing to reframe the transcript and overcome DMD-causing dystrophin gene lesions to generate shorter, partially functional dystrophin isoforms, and is showing promise as a therapy for DMD. Dystrophin gene structure in patients with mild phenotypes can provide templates for potentially functional dystrophin isoforms. However, such mutations downstream of exon 55 are rare, and the probable consequences of AO-induced exon removal in this region are not known. We report that systemic administration of antisense phosphorodiamidate morpholino oligomer-cell penetrating peptide conjugates to wild-type C57BL/10ScSn mice can remove dystrophin exons to generate in vivo dystrophic models for molecular, physiological and pathology evaluation. Exclusion of selected exons within the b dystroglycan and syntrophin binding domains is elucidating the relative importance of these regions to dystrophin function, and may provide guidelines for the development of therapeutic exon skipping strategies.

Background. Antisense oligonucleotides can redirect the pre-mRNA processing of targeted gene transcripts. Therapeutic alternative splicing can be employed excise a selected exon or enhance recognition of an exon normally excluded from the mature mRNA. Aims. Duchenne muscular dystrophy (DMD), the most common and serious form of childhood muscle wasting, arises from protein truncating mutations in DMD that preclude synthesis of a functional protein. We aim to specifically redirect dystrophin pre-mRNA processing so that one or more exons can be excluded from the mature mRNA. Depending upon the DMD mutation, the reading frame can be restored, or intra-exonic protein truncation mutations can be bypassed, allowing an internally deleted but functional dystrophin isoform to be produced. Methods. Targeting dystrophin exon 51 for excision should restore functional dystrophin expression in the most common subset of DMD deletion patients. An extended placebo-controlled study was initiated under the sponsorship of Sarepta therapeutics in Nationwide Children’s Hospital, Columbus Ohio. Results. The trial has now been extended out to over 2 years and clinically significant differences were observed, with treated boys maintaining similar levels of ambulation over the trial period. No serious adverse events have been reported and the trial remains ongoing. Additional oligomers are being designed to address different dystrophin mutations, and new clinical trials should be underway in 2014. Summary. The promising DMD trial results have renewed enthusiasm to pursue splice intervention therapies for other disorders. Spinal muscular atrophy, cystic fibrosis, facioscapulohumeral muscular dystrophy, asthma, Alzheimer’s, Parkinson’s and stroke are just some of the conditions under investigation. An estimated 15% of human mutations induce aberrant splicing and splice switching oligomers may be used as a personalized genetic therapy, regardless of the mutated gene.

Splicing is a fundamental process during the expression of most human gene transcripts, with alternative splicing frequently occurring in a tissue-specific and/or developmental manner to further increase our genetic plasticity. We have shown that antisense oligomers can specifically redirect pre-mRNA processing by either excising a selected exon (blocking positive enhancer elements), or promote retention of an exon normally excluded from the mature mRNA (masking splice silencer motifs). Therapeutic alternative splicing is now in clinical trials to address Duchenne muscular dystrophy (DMD), the most common and serious form of childhood muscle wasting. Protein truncating mutations in the DMD gene that preclude synthesis of a functional protein can be removed during dystrophin pre-mRNA processing with splice switching oligomers. Targeting dystrophin exon 51 for excision will restore functional dystrophin expression in the most common subset of DMD deletion patients (about 10% of DMD boys). Following Phase 1 and 2a trials in the UK, an extended placebo-controlled study was initiated under the sponsorship of Sarepta Therapeutics in Nationwide Children’s Hospital, Columbus, Ohio. The trial has now been underway for nearly 3 years and clinically significant differences were observed, with treated boys maintaining similar levels of ambulation over the trial period. No serious adverse events have been reported and the trial remains ongoing. These promising DMD trial results have renewed enthusiasm to pursue splice intervention therapies for other disorders. Spinal muscular atrophy, cystic fibrosis, myotonic dystrophy, facioscapulohumeral muscular dystrophy, asthma, Alzheimer’s, Parkinson’s and stroke are just some of the conditions currently under investigation in our laboratory. An estimated 15% of human mutations induce aberrant splicing and splice switching oligomers may be used as a personalized genetic therapy, regardless of the mutated gene.

Approximately 20% of boys with Duchenne Muscular Dystrophy will die of dilated cardiomyopathy. The cardiomyopathy is characterised by disrupted structure and function of cardiac muscle cells and reduced energy production. However, the mechanisms responsible for the altered energy metabolism have been poorly understood. We have previously sought to identify the mechanisms for metabolic inhibition in mdx mouse cardiomyopathy. Calcium influx through the L-type Ca2+ channel (also known as the dihydropyridine receptor) in cardiac myocytes is essential for contraction. Calcium is also important for the regulation of mitochondrial function and production of ATP that is required to meet the energy demands of the heart. We have shown that the L-type Ca2+ channel can regulate mitochondrial function and metabolic activity in cardiac myocytes. In mdx heart, the communication between the Ltype Ca2+ channel and the mitochondria is altered as a result of disruption of the cytoskeletal architecture. This contributes to metabolic inhibition in the mdx heart. We demonstrate that treatment of mdx mice with a phophorodiamidate morpholino oligomer, designed to induce skipping of exon 23, “restored” the increase in mitochondrial membrane potential in mdx myocytes after activation of the L-type Ca2+ channel with the dihydropyridine. These results confirm that metabolic inhibition occurs as a result of the absence of dystrophin, and oligomer therapy may be able to normalise metabolic activity and restore contractility in mdx mouse hearts.

Duchenne muscular dystrophy (DMD) is an X-linked, relentlessly progressive muscle wasting disorder resulting from faulty production of the sub sarcolemmal protein, dystrophin. DMD has a predictable course and limited treatment options, with the majority of cases being caused by frame-shifting deletions of one or more of the 79 exons in the dystrophin gene, while deletions that do not disrupt the dystrophin reading frame generally cause the milder allelic disorder, Becker muscular dystrophy (BMD). Antisense oligomer (AO)-mediated splicing manipulation can remove specific exons during transcript processing and overcome DMD-causing dystrophin gene lesions to generate shorter, partially functional BMD-like dystrophin isoforms, and is showing promise as a therapy for DMD. Dystrophin gene structure in BMD patients with less severe phenotypes provides templates for potentially functional dystrophin isoforms. However, such mutations downstream of exon 55 are rare, and the probable consequences of AO-induced exon removal in this region are not known. We report that systemic administration of antisense phosphorodiamidate morpholino oligomer-cell penetrating peptide conjugates to wild-type C57BL/10ScSn mice can remove dystrophin exons to generate DMD- and BMD-like in vivo models for molecular, physiological and pathology evaluation. Exclusion of single exons and in-frame exon blocks, within the β dystroglycan and syntrophin binding domains, is helping to elucidate the relative importance of these regions to dystrophin function, and provide guidelines for the development of therapeutic exon skipping strategies.

Mutations that ablate dystrophin expression lead to Duchenne muscular dystrophy (DMD) an X-linked, relentlessly progressive muscle wasting disorder with a predictable course and limited treatment options. Corticosteroids are effective in stabilizing muscle strength in the short term but do not address the primary etiology of DMD, the absence of dystrophin. The majority of DMD cases are caused by frame-shifting deletions of one or dystrophin exons, while in-frame deletions generally cause the milder allelic disorder, Becker muscular dystrophy (BMD). Antisense oligomer (AO)-mediated splicing manipulation can exclude exons during transcript processing and by-pass DMD-causing mutations to generate shorter, partially functional BMD-like dystrophin isoforms, and is showing promise as a therapy for DMD. Dystrophin genes in selected BMD patients indicate templates for functional dystrophin isoforms, however, in-frame deletions in some regions of the dystrophin gene, particularly downstrean of exon 55 are rare, and the consequences of exon exclusion in this region are unknown. The mdx mouse is a widely used dystrophinopathy model and has a nonsense mutation in dystrophin exon 23. AO induced-excision of this exon from the mRNA removes the mutation without disrupting the reading frame, resulting in functional dystrophin expression and amelioration of the phenotype. We now report that systemic administration of AO combinations to wild-type mice can remove dystrophin exons to generate DMD- and BMD-like dystrophin isoforms for functional evaluation. Assessment of contractile properties of the muscle reveals that some in-frame exon combinations confer near normal function, while others result in muscle susceptible to contraction-induced damage.

Antisense oligomer (AO)-mediated splicing manipulation can remove specific exons during transcript processing, to by-pass DMD-causing dystrophin gene lesions and generate shorter, partially functional BMD-like dystrophin isoforms, and is showing promise as a therapy for DMD. Dystrophin gene structure in mildly affected and asymptomatic BMD patients indicates templates for a number of functional dystrophins, however, in-frame deletions in some regions of the dystrophin gene, particularly 5 of exon 55 are rare and the consequences of exon exclusion in this region are not known. The mdx mouse is a widely used dystrophinopathy model and has a nonsense mutation in dystrophin exon 23. AO induced- excision of this exon from the mRNA removes the mutation without disrupting the reading frame, resulting in functional dystrophin expression and amelioration of the phenotype. We now report that systemic administration of AO combinations to wild-type mice can remove dystrophin exons to generate DMD- and BMD- like dystrophin isoforms for functional evaluation. Assessment of contractile properties of the muscle reveals that some in-frame exon combinations confer near normal function, while others result in muscle susceptible to contraction-induced damage. Furthermore, we show that concurrent administration of prednisolone and AOs improves dystrophin exon skipping and muscle function.