Antisense PMO Found in Dystrophic Dog Model Was Effective in Cells from Exon 7-Deleted DMD Patient

Background

Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD_J) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient''s cells.

Methodology/Principal Findings

We converted fibroblasts of CXMD_J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD_J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species.

Conclusion/Significance

Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.     

Antisense-mediated exon skipping: a versatile tool with therapeutic and research applications

Antisense-mediated modulation of splicing is one of the few fields where antisense oligonucleotides (AONs) have been able to live up to their expectations. In this approach, AONs are implemented to restore cryptic splicing, to change levels of alternatively spliced genes, or, in case of Duchenne muscular dystrophy (DMD), to skip an exon in order to restore a disrupted reading frame. The latter allows the generation of internally deleted, but largely functional, dystrophin proteins and would convert a severe DMD into a milder Becker muscular dystrophy phenotype. In fact, exon skipping is currently one of the most promising therapeutic tools for DMD, and a successful first-in-man trial has recently been completed. In this review the applicability of exon skipping for DMD and other diseases is described. For DMD AONs have been designed for numerous exons, which has given us insight into their mode of action, splicing in general, and splicing of the DMD gene in particular. In addition, retrospective analysis resulted in guidelines for AON design for DMD and most likely other genes as well. This knowledge allows us to optimize therapeutic exon skipping, but also opens up a range of other applications for the exon skipping approach.     

Exon edited dystrophin rods in the hinge 3 region

We have studied the properties of a panel of proteins engineered to be end-products of envisioned exon skipping therapy by antisense oligonucleotides, AONs, directed at exon 51 applied to relevant dystrophin defects causing Duchenne muscular dystrophy, DMD. Exon skipping therapy is a leading therapeutic strategy being investigated for the treatment of this devastating genetic disease. AONs targeting exon 51 have progressed furthest in human clinical trials. Exon 51 skipping is applicable to a variety of dystrophin defects found in different patients. Due to the differences in original defect, the end result of the therapy will be different in each case. An open question is whether these differences will produce significant differences in the dystrophin protein so edited. In this study we have identified differences in the stability, structure and lipid binding properties of these end-product proteins produced by exon 51 skipping repair.     

In vivo comparison of 2′‐O‐methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping

Background

Antisense‐mediated exon skipping is a putative treatment for Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs), the disrupted DMD reading frame is restored, allowing generation of partially functional dystrophin and conversion of a severe Duchenne into a milder Becker muscular dystrophy phenotype. In vivo studies are mainly performed using 2′‐O‐methyl phosphorothioate (2OMePS) or morpholino (PMO) AONs. These compounds were never directly compared.

Methods

mdx and humanized (h)DMD mice were injected intramuscularly and intravenously with short versus long 2OMePS and PMO for mouse exon 23 and human exons 44, 45, 46 and 51.

Results

Intramuscular injection showed that increasing the length of 2OMePS AONs enhanced skipping efficiencies of human exon 45, but decreased efficiency for mouse exon 23. Although PMO induced more mouse exon 23 skipping, PMO and 2OMePS were more comparable for human exons. After intravenous administration, exon skipping and novel protein was shown in the heart with both chemistries. Furthermore, PMO showed lower intramuscular concentrations with higher exon 23 skipping levels compared to 2OMePS, which may be due to sequestration in the extracellular matrix. Finally, two mismatches rendered 2OMePS but not PMO AONs nearly ineffective.

Conclusions

The results obtained in the present study indicate that increasing AON length improves skipping efficiency in some but not all cases. It is feasible to induce exon skipping and dystrophin restoration in the heart after injection of 2OMePS and unconjugated PMO. Furthermore, differences in efficiency between PMO and 2OMePS appear to be sequence and not chemistry dependent. Finally, the results indicate that PMOs may be less sequence specific than 2OMePS. Copyright © 2009 John Wiley & Sons, Ltd.     

Frameshift deletions of exons 3-7 and revertant fibers in Duchenne muscular dystrophy: mechanisms of dystrophin production.

Duchenne muscular dystrophy (DMD) patients with mutations that disrupt the translational reading frame produce little or no dystrophin. Two exceptions are the deletion of exons 3-7 and the occurrence of rare dystrophin-positive fibers (revertant fibers) in muscle of DMD patients. Antibodies directed against the amino-terminus and the 5' end of exon 8 did not detect dystrophin in muscle from patients who have a deletion of exons 3-7. However, in all cases, dystrophin was detected with an antibody directed against the 3' end of exon 8. The most likely method of dystrophin production in these cases is initiation at a new start codon in exon 8. We also studied two patients who have revertant fibers: one had an inherited duplication of exons 5-7, which, on immunostaining, showed two types of revertant fibers; and the second patient had a 2-bp nonsense mutation in exon 51, which creates a cryptic splice site. An in-frame mRNA that uses this splice site in exon 51 was detected. Immunostaining demonstrated the presence of the 3' end of exon 51, which is in agreement with the use of this mRNA in revertant fibers. The most likely method of dystrophin production in these fibers is a second mutation that restores the reading frame.     

Extensive and prolonged restoration of dystrophin expression with vivo-morpholino-mediated multiple exon skipping in dystrophic dogs

Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6-8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD.     

Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology

For the majority of Duchenne muscular dystrophy (DMD) mutations, antisense oligonucleotide (AON)-mediated exon skipping has the potential to restore a functional protein. Here we show that weekly intravenous injections of morpholino phosphorodiamidate (morpholino) AONs induce expression of functional levels of dystrophin in body-wide skeletal muscles of the dystrophic mdx mouse, with resulting improvement in muscle function. Although the level of dystrophin expression achieved varies considerably between muscles, antisense therapy may provide a realistic hope for the treatment of a majority of individuals with DMD.     

miR-31 modulates dystrophin expression: new implications for Duchenne muscular dystrophy therapy

Duchenne muscular dystrophy (DMD)--which is caused by mutations in the dystrophin gene-is one of the most severe myopathies. Among therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional messenger RNA. Here, we report the identification of a microRNA--miR-31--that represses dystrophin expression by targeting its 3' untranslated region. In human DMD myoblasts treated with exon skipping, we demonstrate that miR-31 inhibition increases dystrophin rescue. These results indicate that interfering with miR-31 activity can provide an ameliorating strategy for those DMD therapies that are aimed at efficiently recovering dystrophin synthesis.     

Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intramuscular and intra-arterial delivery of genetically corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recovery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.     

Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy.

The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random "libraries" of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25-60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1-41, and we now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes.     

Analysis of a dystrophin gene deletion by amplification of mRNA isolated from DMD myotubes cultured in vitro

The most frequent causes for the X-linked muscular dystrophy of the allelic Duchenne (DMD) or Becker (BMD) type are partial deletions of the dystrophin gene. These mutations are accompanied either by disrupted or by preserved translational reading frames in mRNAs derived from the deleted genes. As a rule, the reading frame is destroyed in the more severe DMD, whereas it is preserved in the less severe BMD (M. Koenig et al., 1989, Am. J. Hum. Genet. 45, 498-506). We have analyzed in detail a deletion that was detected in a fetus at risk of DMD. The analysis of this mutation included the delineation of the altered subregion in the dystrophin mRNA. mRNA was isolated from myotubes derived from embryonic DMD myoblasts propagated in vitro. This study was based on enzymatic amplification by the polymerase chain reaction (PCR) of dystrophin mRNA and direct sequencing of the amplified cDNA. Exons 47 to 50 were found to be missing in the mRNA. The splicing of exon 46 to exon 51 resulted in a reading frameshift, indicating that this mutation is likely to be responsible for a DMD type of dystrophy. The clinical diagnosis of DMD for a 10-year-old patient in this family was compatible with the "reading frame" assumption.     

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.     

Dystromirs as Serum Biomarkers for Monitoring the Disease Severity in Duchenne Muscular Dystrophy

Duchenne muscular Dystrophy (DMD) is an inherited disease caused by mutations in the dystrophin gene that disrupt the open reading frame, while in frame mutations result in Becker muscular dystrophy (BMD). Ullrich congenital muscular dystrophy (UCMD) is due to mutations affecting collagen VI genes. Specific muscle miRNAs (dystromirs) are potential non-invasive biomarkers for monitoring the outcome of therapeutic interventions and disease progression. We quantified miR-1, miR-133a,b, miR-206 and miR-31 in serum from patients with DMD, BMD, UCMD and healthy controls. MiR-1, miR-133a,b and miR-206 were upregulated in DMD, but unchanged in UCMD compared to controls. Milder DMD patients had higher levels of dystromirs than more severely affected patients. Patients with low forced vital capacity (FVC) values, indicating respiratory muscle weakness, had low levels of serum miR-1 and miR-133b. There was no significant difference in the level of the dystromirs in BMD compared to controls.We also assessed the effect of dystrophin restoration on the expression of the five dystromirs in serum of DMD patients treated systemically for 12 weeks with antisense oligomer eteplirsen that induces skipping of exon 51 in the dystrophin gene. The dystromirs were also analysed in muscle biopsies of DMD patients included in a single dose intramuscular eteplirsen clinical trial. Our analysis detected a trend towards normalization of these miRNA between the pre- and post-treatment samples of the systemic trial, which however failed to reach statistical significance. This could possibly be due to the small number of patients and the short duration of these clinical trials.Although longer term studies are needed to clarify the relationship between dystrophin restoration following therapeutic intervention and the level of circulating miRNAs, our results indicate that miR-1 and miR-133 can be considered as exploratory biomarkers for monitoring the progression of muscle weakness and indirectly the remaining muscle mass in DMD.     

Gene therapy for muscle disease

The molecular mechanisms of Duchenne muscular dystrophy (DMD) have been extensively investigated since the discovery of the dystrophin gene in 1986. Nonetheless, there is currently no effective treatment for DMD. Recent reports, however, indicate that adenoassociated viral (AAV) vector-mediated transfer of a functional dystrophin cDNA into the affected muscle is a promising strategy. In addition, antisense-mediated exon skipping technology has been emerging as another promising approach to restore dystrophin expression in DMD muscle. Ongoing clinical trials show restoration of dystrophin in DMD patients without serious side effects. Here, we summarize the recent progress in gene therapy, with an emphasis on exon skipping for DMD.     

Duchenne and Becker muscular dystrophy: from gene diagnosis to molecular therapy

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked muscular dystrophies. The isolation of the defective gene in DMD/BMD has led to a better understanding of the disease process and has promoted studies regarding the application of molecular therapy. The purpose of this review is to present the progress made in this area of research with particular reference to dystrophin Kobe. Based on the results from the molecular analysis of dystrophin Kobe, we propose a novel molecular therapeutic method for DMD in which antisense oligonucleotides transform DMD into a milder phenotype by inducing exon skipping. In addition, current proposals for the molecular therapy of DMD are discussed.