Restore Dystrophin Expression Research Articles

P.142Very low residual dystrophin levels mitigate dystrophinopathy towards Becker muscular dystrophy

Novel therapy approaches for Duchenne muscular dystrophy aim at restoring dystrophin expression. However, the minimal dystrophin restoration level required to achieve clinical benefit remains unknown. Residual dystrophin expression can be observed in certain patients carrying DMD intronic (including splice sites) and nonsense mutations in exons for which deletion leads to in-frame transcripts. To determine whether expression of very low dystrophin levels (<5%) can mitigate disease severity, residual dystrophin expression was correlated with disease severity of patients with these DMD mutations. Seventy-seven patients were identified using the UMD-DMD-France database. Clinical data were obtained from medical care centres. Residual dystrophin expression was semi-quantitatively determined on Western Blots. Time-to-event analysis was performed for age of loss-of-ambulation as well as for respiratory, cardiac and orthopaedic endpoints and life expectancy. Patients with <5% dystrophin expression ambulated longer (n=33) than patients without dystrophin (n=44), (p<0.001). Furthermore, patients with <5% dystrophin had longer survival (p<0.001), delayed decline in Left Ventricular Ejection Function [LVEF] <55% (p<0.001), delayed decline in vital capacity [CV] <50% (p<0.001) and in CV<20% (p<0.001), later start of non-invasive ventilation (p<0.001), tracheotomy (p<0.001) and arthrodesis (p<0.001). In conclusion, very low residual dystrophin expression is sufficient to shift the natural history of dystrophinopathy towards the milder Becker phenotype. These data suggest that restoration of low dystrophin levels might be sufficient to achieve long-term clinical benefit from novel therapeutic strategies. Results will be discussed in the light of the limited number of study subjects, time of observation and employed western blot methodology.

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmdmdx mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.

Autoimmune response and its long-term consequences after exon-skipping therapy in a Duchenne muscular dystrophy mouse model.

The progress of antisense-based therapies using first generation Morpholino oligonucleotides for Duchenne muscular dystrophy (DMD) is expected to partially restore dystrophin expression and may prolong the lifespan of DMD patients. In a recent issue of The Journal of Pathology, a sophisticated study by Vila et al used a dystrophic mouse model of DMD to demonstrate that Morpholino-induced exon skipping induced dystrophin expression in skeletal muscle and stimulated cell mediated and humoral responses to dystrophin. The study highlights the need to further investigate the autoimmune response against de novo synthesised truncated dystrophin protein and its long-term consequences after exon-skipping therapy for DMD. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Dystrophin deficiency leads to dysfunctional glutamate clearance in iPSC derived astrocytes

Duchenne muscular dystrophy (DMD) results, beside muscle degeneration in cognitive defects. As neuronal function is supported by astrocytes, which express dystrophin, we hypothesized that loss of dystrophin from DMD astrocytes might contribute to these cognitive defects. We generated cortical neuronal and astrocytic progeny from induced pluripotent stem cells (PSC) from six DMD subjects carrying different mutations and several unaffected PSC lines. DMD astrocytes displayed cytoskeletal abnormalities, defects in Ca+2 homeostasis and nitric oxide signaling. In addition, defects in glutamate clearance were identified in DMD PSC-derived astrocytes; these deficits were related to a decreased neurite outgrowth and hyperexcitability of neurons derived from healthy PSC. Read-through molecule restored dystrophin expression in DMD PSC-derived astrocytes harboring a premature stop codon mutation, corrected the defective astrocyte glutamate clearance and prevented associated neurotoxicity. We propose a role for dystrophin deficiency in defective astroglial glutamate homeostasis which initiates defects in neuronal development.

Life-Long AAV-Mediated CRISPR Genome Editing in Dystrophic Heart Improves Cardiomyopathy without Causing Serious Lesions in mdx Mice.

Previous studies from others and us have demonstrated that CRISPR genome editing could offer a promising therapeutic strategy to restore dystrophin expression and function in the skeletal muscle and heart of Duchenne muscular dystrophy (DMD) mouse models. However, the long-term efficacy and safety of CRISPR genome-editing therapy for DMD has not been well established. We packaged both SaCas9 and guide RNA (gRNA) together into one AAVrh.74 vector, injected two such vectors (targeting intron 20 and intron 23, respectively) into mdx pups at day 3 and evaluated the mice at 19months. We found that AAVrh.74-mediated life-long CRISPR genome editing in mdx mice restored dystrophin expression and improved cardiac function without inducing serious adverse effects. PCR analysis and targeted deep sequencing showed that the DSBs were mainly repaired by the precise ligation of the two cut sites. Serological and histological examination of major vital organs did not reveal any signs of tumor development or other deleterious defects arising from CRISPR genome editing. These results support that invivo CRISPR genome editing could be developed as a safe therapeutic treatment for DMD and potentially other diseases.

Morpholino-induced exon skipping stimulates cell-mediated and humoral responses to dystrophin in mdx mice.

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded 'revertant' dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mutation-Based Therapeutic Strategies for Duchenne Muscular Dystrophy: From Genetic Diagnosis to Therapy.

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked muscle disorders caused by mutations of the DMD gene, which encodes the subsarcolemmal protein dystrophin. In DMD, dystrophin is not expressed due to a disruption in the reading frame of the DMD gene, resulting in a severe phenotype. Becker muscular dystrophy exhibits a milder phenotype, having mutations that maintain the reading frame and allow for the production of truncated dystrophin. To date, various therapeutic approaches for DMD have been extensively developed. However, the pathomechanism is quite complex despite it being a single gene disorder, and dystrophin is expressed not only in a large amount of skeletal muscle but also in cardiac, vascular, intestinal smooth muscle, and nervous system tissue. Thus, the most appropriate therapy would be complementation or restoration of dystrophin expression, such as gene therapy using viral vectors, readthrough therapy, or exon skipping therapy. Among them, exon skipping therapy with antisense oligonucleotides can restore the reading frame and yield the conversion of a severe phenotype to one that is mild. In this paper, I present the significance of molecular diagnosis and the development of mutation-based therapeutic strategies to complement or restore dystrophin expression.

Restoring Dystrophin Expression in Duchenne Muscular Dystrophy: Current Status of Therapeutic Approaches.

Duchenne muscular dystrophy (DMD), a rare genetic disorder characterized by progressive muscle weakness, is caused by the absence or a decreased amount of the muscle cytoskeletal protein dystrophin. Currently, several therapeutic approaches to cure DMD are being investigated, which can be categorized into two groups: therapies that aim to restore dystrophin expression, and those that aim to compensate for the lack of dystrophin. Therapies that restore dystrophin expression include read-through therapy, exon skipping, vector-mediated gene therapy, and cell therapy. Of these approaches, the most advanced are the read-through and exon skipping therapies. In 2014, ataluren, a drug that can promote ribosomal read-through of mRNA containing a premature stop codon, was conditionally approved in Europe. In 2016, eteplirsen, a morpholino-based chemical capable of skipping exon 51 in premature mRNA, received conditional approval in the USA. Clinical trials on vector-mediated gene therapy carrying micro- and mini- dystrophin are underway. More innovative therapeutic approaches include CRISPR/Cas9-based genome editing and stem cell-based cell therapies. Here we review the current status of therapeutic approaches for DMD, focusing on therapeutic approaches that can restore dystrophin.

CRISPR alleviates muscular dystrophy in dogs.

Widespread editing of the mutated DMD gene by CRISPR–Cas9, systemically delivered via an adeno-associated virus, restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

Efficacy of Multi-exon Skipping Treatment in Duchenne Muscular Dystrophy Dog Model Neonates

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

Gene editing and muscular dystrophy

Muscle Disease Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness and a shortened life span. The disease is caused by mutations that reduce or prevent expression of dystrophin, an essential structural protein in skeletal and heart muscle. The gene editing technology CRISPR-Cas9 can correct disease-causing mutations and has yielded promising results in mouse models of DMD. In a small, short-term study, Amoasii et al. tested this strategy in a dog model of DMD that exhibits many features of the human disease. Intramuscular or systemic delivery of the gene editing components resulted in a substantial increase in dystrophin protein levels in skeletal and heart muscle. Restoration of dystrophin expression was accompanied by improved muscle histology. Science , this issue p. [86][1] [1]: /lookup/doi/10.1126/science.aau1549

Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational "hotspot" in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD.

Exosome-Derived Dystrophin from Allograft Myogenic Progenitors Improves Cardiac Function in Duchenne Muscular Dystrophic Mice.

Progressive cardiomyocyte loss in Duchenne muscular dystrophy (DMD) leads to cardiac fibrosis, cardiomyopathy, and eventually heart failure. In the present study, we observed that myogenic progenitor cells (MPC) carry mRNA for the dystrophin gene. We tested whether cardiac function can be improved in DMD by allograft transplantation of MPC-derived exosomes (MPC-Exo) into the heart to restore dystrophin protein expression. Exo from C2C12 cells (an MPC cell line) or vehicle were delivered locally into the hearts of MDX mice. After 2days of treatment, we observed that MPC-Exo restored dystrophin expression in the hearts of MDX mice, which correlated with improved myocardial function in dystrophin-deficient MDX mouse hearts. In conclusion, this study demonstrated that allogeneic WT-MPC-Exo transplantation transiently restored dystrophin gene expression and improved cardiac function in MDX mice, suggesting that allogenic exosomal delivery may serve as an alternative treatment for cardiomyopathy of DMD.

CRISPR-Induced Deletion with SaCas9 Restores Dystrophin Expression in Dystrophic Models InVitro and InVivo.

Duchenne muscular dystrophy (DMD), a severe hereditary disease affecting 1 in 3,500 boys, mainly results from the deletion of exon(s), leading to a reading frameshift of the DMD gene that abrogates dystrophin protein synthesis. Pairs of sgRNAs for the Cas9 of Staphylococcus aureus were meticulously chosen to restore a normal reading frame and also produce a dystrophin protein with normally phased spectrin-like repeats (SLRs), which is not usually obtained by skipping or by deletion of complete exons. This can, however, be obtained in rare instances where the exon and intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion) are at similar positions in the SLR. We used pairs of sgRNAs targeting exons 47 and 58, and a normal reading frame was restored in myoblasts derived from muscle biopsies of 4 DMD patients with different exon deletions. Restoration of the DMD reading frame and restoration of dystrophin expression were also obtained invivo in the heart of the del52hDMD/mdx. Our results provide a proof of principle that SaCas9 could be used to edit the human DMD gene and could be considered for further development of a therapy for DMD.

Dystrophin Expressing Chimeric (DEC) Human Cells Provide a Potential Therapy for Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a progressive and lethal disease caused by mutations of the dystrophin gene. Currently no cure exists. Stem cell therapies targeting DMD are challenged by limited engraftment and rejection despite the use of immunosuppression. There is an urgent need to introduce new stem cell-based therapies that exhibit low allogenic profiles and improved cell engraftment. In this proof-of-concept study, we develop and test a new human stem cell-based approach to increase engraftment, limit rejection, and restore dystrophin expression in the mdx/scid mouse model of DMD. We introduce two Dystrophin Expressing Chimeric (DEC) cell lines created by ex vivo fusion of human myoblasts (MB) derived from two normal donors (MBN1/MBN2), and normal and DMD donors (MBN/MBDMD). The efficacy of fusion was confirmed by flow cytometry and confocal microscopy based on donor cell fluorescent labeling (PKH26/PKH67). In vitro, DEC displayed phenotype and genotype of donor parent cells, expressed dystrophin, and maintained proliferation and myogenic differentiation. In vivo, local delivery of both DEC lines (0.5 × 106) restored dystrophin expression (17.27%±8.05—MBN1/MBN2 and 23.79%±3.82—MBN/MBDMD) which correlated with significant improvement of muscle force, contraction and tolerance to fatigue at 90 days after DEC transplant to the gastrocnemius muscles (GM) of dystrophin-deficient mdx/scid mice. This study establishes DEC as a potential therapy for DMD and other types of muscular dystrophies.

Low dystrophin levels are insufficient to normalize the neuromuscular synaptic abnormalities of mdx mice

Dystrophin is a sub-sarcolemmal component of skeletal muscle fibres and is enriched at the postsynaptic membrane of the neuromuscular junction (NMJ). In the mdx mouse, dystrophin absence not only causes muscle damage but also mild synaptic dysfunctions and clear morphological aberrations at NMJs. In particular, reduction of postsynaptic sensitivity for the neurotransmitter acetylcholine and extra exhaustion of presynaptic acetylcholine release during intense synaptic activity exists. Current experimental therapeutic approaches in Duchenne muscular dystrophy aim to restore dystrophin expression. An important question is what dystrophin levels are needed to improve muscle function. Recent experimental and clinical studies suggested that levels as low as a few percent of normal can be beneficial. Similarly, it is of interest to know how dystrophin levels relate to NMJ function and morphology. We investigated NMJs of a series of mdx-XistΔhs mice, which expressed dystrophin between ~2% and 19% of normal. Most functional and morphological NMJ parameters of these mice remained comparable to mdx. On the other hand, mdx+/- mice (expressing ~50% dystrophin) showed normal NMJ features. Thus, the minimal dystrophin level required for normal NMJ function and morphology lies between 19% and 50% of normal when expression of dystrophin is not uniform.

Myoediting for Duchenne muscular dystrophy

Genetics![Figure][1] A new technology brings hope in the fight against DMD. CREDIT: DISABILITY/SCIENCE SOURCE Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that results in progressive weakening and muscle loss. Long et al. used CRISPR-Cas9 genome editing to correct the hotspot mutations that disrupt expression of the dystrophin gene in DMD. So-called myoediting abolished RNA splice sites and allowed the removal of the most common mutations. The approach successfully restored dystrophin expression in patient-derived induced pluripotent stem cells and restored mechanical force contraction in derived cardiomyocytes to near normal. Sci. Adv. 10.1126/sciadv.aap9004 (2018). [1]: pending:yes

In Vitro Multiexon Skipping by Antisense PMOs in Dystrophic Dog and Exon 7-Deleted DMD Patient.

Antisense oligonucleotide induced exon skipping emerges as a promising therapeutic strategy for patients suffering from a devastating muscle disorder Duchenne muscular dystrophy (DMD). Systemic administration of antisense phosphorodiamidate morpholino oligomers (PMOs) targeting exons 6 and 8 in dystrophin mRNA of the canine X-linked muscular dystrophy model in Japan (CXMDJ) that lacks exon 7, restored dystrophin expression throughout skeletal muscle and ameliorated skeletal muscle pathology and function. However, the antisense PMO regime used in CXMDJ could not be considered for a direct application to DMD patients so far, because this type of mutation is quite rare. We have identified a DMD patient with an exon 7 deletion; and tried a direct translation of the antisense PMOs used in dog models to the DMD patient's cells. We converted fibroblasts obtained from CXMDJ dogs and from the DMD patient to myotubes by MyoD transduction using fluorescence-activated cell sorting (FACS). We subsequently designed antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 and administered them as a cocktail to the in vitro generated dog or human myotubes. In both cases, we observed comparable skipping efficacy of exons 6 and 8 and restoration of dystrophin protein. The accompanying skipping of exon 9, which does not alter the reading frame, varied according to the cell origin. The antisense PMOs originally administered to the CXMDJ dog model were capable of inducing multi-exon skipping of the dystrophin gene on the FACS-aided MyoD-transduced fibroblasts derived from an exon 7-deleted DMD patient. These data support the suitability of dog as a laboratory model for DMD because the similarity of dystrophin sequences allowed a successful translation of the dog's PMOs to DMD patients cells.

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing.

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.