A.M. Adams

Rational design of antisense oligomers to induce dystrophin exon skipping

Duchenne muscular dystrophy (DMD), one of the most severe neuromuscular disorders of childhood, is caused by the absence of a functional dystrophin. Antisense oligomer (AO) induced exon skipping is being investigated to restore functional dystrophin expression in models of muscular dystrophy and DMD patients. One of the major challenges will be in the development of clinically relevant oligomers and exon skipping strategies to address many different mutations. Various models, including cell-free extracts, cells transfected with artificial constructs, or mice with a human transgene, have been proposed as tools to facilitate oligomer design. Despite strong sequence homology between the human and mouse dystrophin genes, directing an oligomer to the same motifs in both species does not always induce comparable exon skipping. We report substantially different levels of exon skipping induced in normal and dystrophic human myogenic cell lines and propose that animal models or artificial assay systems useful in initial studies may be of limited relevance in designing the most efficient compounds to induce targeted skipping of human dystrophin exons for therapeutic outcomes.

Impaired functional communication between the L-type calcium channel and mitochondria contributes to metabolic inhibition in the mdx heart

Significance Duchenne muscular dystrophy (DMD) is a fatal X-linked disease that results in cardiomyopathy and heart failure. The cardiomyopathy is characterized by cytoskeletal protein disarray, contractile dysfunction, and reduced energy production. The mechanisms for altered energy metabolism are not yet fully clarified. The L-type Ca2+ channel regulates excitation and contraction in the heart, and can regulate mitochondrial function via the movement of cytoskeletal proteins. Here, we find that myocytes from the murine model of DMD (mdx) exhibit impaired communication between the L-type Ca2+ channel and the mitochondria that results in poor energy production. Morpholino oligomer therapy targeting dystrophin or block of the mitochondrial voltage-dependent anion channel (VDAC) “rescues” metabolic function, indicating that impaired communication between the L-type Ca2+ channel and VDAC contributes to the cardiomyopathy. Duchenne muscular dystrophy is a fatal X-linked disease characterized by the absence of dystrophin. Approximately 20% of boys will die of dilated cardiomyopathy that is associated with cytoskeletal protein disarray, contractile dysfunction, and reduced energy production. However, the mechanisms for altered energy metabolism are not yet fully clarified. Calcium influx through the L-type Ca2+ channel is critical for maintaining cardiac excitation and contraction. The L-type Ca2+ channel also regulates mitochondrial function and metabolic activity via transmission of movement of the auxiliary beta subunit through intermediate filament proteins. Here, we find that activation of the L-type Ca2+ channel is unable to induce increases in mitochondrial membrane potential and metabolic activity in intact cardiac myocytes from the murine model of Duchenne muscular dystrophy (mdx) despite robust increases recorded in wt myocytes. Treatment of mdx mice with morpholino oligomers to induce exon skipping of dystrophin exon 23 (that results in functional dystrophin accumulation) or application of a peptide that resulted in block of voltage-dependent anion channel (VDAC) “rescued” mitochondrial membrane potential and metabolic activity in mdx myocytes. The mitochondrial VDAC coimmunoprecipitated with the L-type Ca2+ channel. We conclude that the absence of dystrophin in the mdx ventricular myocyte leads to impaired functional communication between the L-type Ca2+ channel and mitochondrial VDAC. This appears to contribute to metabolic inhibition. These findings provide new mechanistic and functional insight into cardiomyopathy associated with Duchenne muscular dystrophy.

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.

Multiple exon skipping strategies to by-pass dystrophin mutations

Manipulation of dystrophin pre-mRNA processing offers the potential to overcome mutations in the dystrophin gene that would otherwise lead to Duchenne muscular dystrophy. Dystrophin mutations will require the removal of one or more exons to restore the reading frame and in some cases, multiple exon skipping strategies exist to restore dystrophin expression. However, for some small intra-exonic mutations, a third strategy, not applicable to whole exon deletions, may be possible. The removal of only one frame-shifting exon flanking the mutation-carrying exon may restore the reading frame and allow synthesis of a functional dystrophin isoform, providing that no premature termination codons are encountered. For these mutations, the removal of only one exon offers a simpler, cheaper and more feasible alternative approach to the dual exon skipping that would otherwise be considered. We present strategies to by-pass intra-exonic dystrophin mutations that clearly demonstrate the importance of tailoring exon skipping strategies to specific patient mutations.

Mismatched single stranded antisense oligonucleotides can induce efficient dystrophin splice switching

BackgroundAntisense oligomer induced exon skipping aims to reduce the severity of Duchenne muscular dystrophy by redirecting splicing during pre-RNA processing such that the causative mutation is by-passed and a shorter but partially functional Becker muscular dystrophy-like dystrophin isoform is produced. Normal exons are generally targeted to restore the dystrophin reading frame however, an appreciable subset of dystrophin mutations are intra-exonic and therefore have the potential to compromise oligomer efficiency, necessitating personalised oligomer design for some patients. Although antisense oligomers are easily personalised, it remains unclear whether all patient polymorphisms within antisense oligomer target sequences will require the costly process of producing and validating patient specific compounds.MethodsHere we report preclinical testing of a panel of splice switching antisense oligomers, designed to excise exon 25 from the dystrophin transcript, in normal and dystrophic patient cells. These patient cells harbour a single base insertion in exon 25 that lies within the target sequence of an oligomer shown to be effective at removing exon 25.ResultsIt was anticipated that such a mutation would compromise oligomer binding and efficiency. However, we show that, despite the mismatch an oligomer, designed and optimised to excise exon 25 from the normal dystrophin mRNA, removes the mutated exon 25 more efficiently than the mutation-specific oligomer.ConclusionThis raises the possibility that mismatched AOs could still be therapeutically applicable in some cases, negating the necessity to produce patient-specific compounds.

Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies

Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes.

Antisense oligonucleotide induction of progerin in human myogenic cells

We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA) transcript in human myogenic cells. The progerin transcript (LMNA Δ150) lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the ‘natural’ ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2′-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model to investigate the role of progerin in premature muscle ageing.

Rational Design of Short Locked Nucleic Acid-Modified 2′-O-Methyl Antisense Oligonucleotides for Efficient Exon-Skipping In Vitro

Locked nucleic acid is a prominent nucleic acid analog with unprecedented target binding affinity to cDNA and RNA oligonucleotides and shows remarkable stability against nuclease degradation. Incorporation of locked nucleic acid nucleotides into an antisense oligonucleotide (AO) sequence can reduce the length required without compromising the efficacy. In this study, we synthesized a series of systematically truncated locked nucleic acid-modified 2′-O-methyl AOs on a phosphorothioate (PS) backbone that were designed to induce skipping exon 23 from the dystrophin transcript in H-2Kb-tsA58 mdx mouse myotubes in vitro. The results clearly demonstrated that shorter AOs (16- to 14-mer) containing locked nucleic acid nucleotides efficiently induced dystrophin exon 23 skipping compared with the corresponding 2′-O-methyl AOs. Our remarkable findings contribute significantly to the existing knowledge about the designing of short LNA-modified oligonucleotides for exon-skipping applications, which will help reduce the cost of exon-skipping AOs and potential toxicities, particularly the 2′-OMe-based oligos, by further reducing the length of AOs.

A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell’s endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple “Phylomer” CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne’s muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.

A morpholino oligomer therapy regime that restores mitochondrial function and prevents mdx cardiomyopathy

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