K. Greer

Targeted Exon Skipping to Correct Exon Duplications in the Dystrophin Gene

Duchenne muscular dystrophy is a severe muscle-wasting disease caused by mutations in the dystrophin gene that ablate functional protein expression. Although exonic deletions are the most common Duchenne muscular dystrophy lesion, duplications account for 10–15% of reported disease-causing mutations, and exon 2 is the most commonly duplicated exon. Here, we describe the in vitro evaluation of phosphorodiamidate morpholino oligomers coupled to a cell-penetrating peptide and 2′-O-methyl phosphorothioate oligonucleotides, using three distinct strategies to reframe the dystrophin transcript in patient cells carrying an exon 2 duplication. Differences in exon-skipping efficiencies in vitro were observed between oligomer analogues of the same sequence, with the phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide proving the most effective. Differences in exon 2 excision efficiency between normal and exon 2 duplication cells, were apparent, indicating that exon context influences oligomer-induced splice switching. Skipping of a single copy of exon 2 was induced in the cells carrying an exon 2 duplication, the simplest strategy to restore the reading frame and generate a normal dystrophin transcript. In contrast, multiexon skipping of exons 2–7 to generate a Becker muscular dystrophy-like dystrophin transcript was more challenging and could only be induced efficiently with the phosphorodiamidate morpholino oligomer chemistry.

Targeted Exon Skipping to Address “Leaky” Mutations in the Dystrophin Gene

Protein-truncating mutations in the dystrophin gene lead to the progressive muscle wasting disorder Duchenne muscular dystrophy, whereas in-frame deletions typically manifest as the milder allelic condition, Becker muscular dystrophy. Antisense oligomer-induced exon skipping can modify dystrophin gene expression so that a disease-associated dystrophin pre-mRNA is processed into a Becker muscular dystrophy-like mature transcript. Despite genomic deletions that may encompass hundreds of kilobases of the gene, some dystrophin mutations appear “leaky”, and low levels of high molecular weight, and presumably semi-functional, dystrophin are produced. A likely causative mechanism is endogenous exon skipping, and Duchenne individuals with higher baseline levels of dystrophin may respond more efficiently to the administration of splice-switching antisense oligomers. We optimized excision of exons 8 and 9 in normal human myoblasts, and evaluated several oligomers in cells from eight Duchenne muscular dystrophy patients with deletions in a known “leaky” region of the dystrophin gene. Inter-patient variation in response to antisense oligomer induced skipping in vitro appeared minimal. We describe oligomers targeting exon 8, that unequivocally increase dystrophin above baseline in vitro, and propose that patients with leaky mutations are ideally suited for participation in antisense oligomer mediated splice-switching clinical studies.

Dystrophin Isoform Induction In Vivo by Antisense-mediated Alternative Splicing

Antisense oligomer-induced manipulation of dystrophin pre-mRNA processing can remove exons carrying mutations, or exclude exons flanking frameshifting mutations, and restore dystrophin expression in dystrophinopathy models and in Duchenne muscular dystrophy (DMD) patients. Splice intervention can also be used to manipulate the normal dystrophin pre-mRNA processing and ablate dystrophin expression in wild-type mice, with signs of pathology being induced in selected muscles within 4 weeks of commencing treatment. The disruption of normal dystrophin pre-mRNA processing to alter the reading frame can be very efficient and offers an alternative mechanism to RNA silencing for gene suppression. In addition, it is possible to remove in-frame exon blocks from the DMD gene transcript and induce specific dystrophin isoforms that retain partial functionality, without having to generate transgenic animal models. Specific exon removal to yield in-frame dystrophin transcripts will facilitate mapping of functional protein domains, based upon exon boundaries, and will be particularly relevant where there is either limited, or conflicting information as to the consequences of in-frame dystrophin exon deletions on the clinical severity and progression of the dystrophinopathy.

Pseudoexon activation increases phenotype severity in a Becker muscular dystrophy patient

We report a dystrophinopathy patient with an in‐frame deletion of DMD exons 45–47, and therefore a genetic diagnosis of Becker muscular dystrophy, who presented with a more severe than expected phenotype. Analysis of the patient DMD mRNA revealed an 82 bp pseudoexon, derived from intron 44, that disrupts the reading frame and is expected to yield a nonfunctional dystrophin. Since the sequence of the pseudoexon and canonical splice sites does not differ from the reference sequence, we concluded that the genomic rearrangement promoted recognition of the pseudoexon, causing a severe dystrophic phenotype. We characterized the deletion breakpoints and identified motifs that might influence selection of the pseudoexon. We concluded that the donor splice site was strengthened by juxtaposition of intron 47, and loss of intron 44 silencer elements, normally located downstream of the pseudoexon donor splice site, further enhanced pseudoexon selection and inclusion in the DMD transcript in this patient.

G.P.78 Dystrophin isoforms with incomplete β dystroglycan and syntrophin binding domains retain partial function

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.

P4.31 Transient mouse models for the preclinical evaluation of therapeutic dystrophin 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.

Splice-switching as a treament for duchenne muscular dystrophy

Antisense oligomer manipulation of pre-mRNA splicing can be used to remove exons carrying premature stop codons or to restore the reading frame around frame-shifting mutations. Phase 1 clinical trials have demonstrated that antisense oligomer-mediated splicing manipulation can restore dystro-phin expression in muscle in a subset of Duchenne muscular dystrophy (DMD) patients. The complexities of dystrophin gene expression and widespread distribution of the protein have presented major challenges to gene and cell therapies for DMD. However, the size of the dystrophin gene, and the fact that not all of the 79 exons are necessary to encode a protein with at least partial function, make DMD amenable to splice switching intervention. While dystrophin deletions are clustered in two hotspots, non-deletion mutations are spread throughout the gene. Although 10 oligomers will be required to restore the reading-frame around the more common deletions, numerous compounds are required to by-pass the many different mutations that cause DMD. We have developed compounds to remove each of the dystrophin exons and are currently optimising preparations to remove in-frame blocks of exons to optimise induced dystrophin isoform function. The next challenge will be to extend the application of antisense oligomers to other conditions that may benefit from splice switching.

T.P.1.06 Induced non-productive splicing to study muscle gene expression

RNA silencing has been applied to suppress gene expression, with varying degrees of specificity and efficiency reported. Endogenous alternative splicing can regulate gene expression through a process called Regulated Unproductive Splicing and Translation (RUST), by either incorporating an exon carrying a nonsense mutation, or excising an exon to disrupt the reading frame. As a result, the mature gene transcripts cannot be translated into functional products. We show that it is possible to efficiently disrupt the normal dystrophin mRNA reading frame and ablate dystrophin expression. Total suppression of dystrophin gene expression can be induced and maintained for several weeks in vivo, and a severe dystrophic pathology observed within 4 weeks of commencing treatment in neonatal normal mice. This approach to gene down-regulation is very efficient and specific. Disruption of gene expression by selected exon exclusion could be applied to many different genes, and offers the opportunity to induce transient mouse models to study the consequences of gene suppression in vivo. In addition, selected exon removal to yield in-frame transcripts can allow mapping of functional protein domains, based upon exon boundaries, and provide a possible alternative to transgenic mouse models for the study of muscle gene expression. We are applying this approach to defining dystrophin functional domains, to optimise exon-skipping strategies for the treatment of Duchenne Muscular Dystrophy.