Restore Dystrophin Expression Research Articles

Developing DMD therapeutics: a review of the effectiveness of small molecules, stop-codon readthrough, dystrophin gene replacement, and exon-skipping therapies

ABSTRACT Introduction Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the dystrophin (DMD) gene. Most patients die from respiratory failure or cardiomyopathy. There are significant unmet needs for treatments for DMD as the standard of care is principally limited to symptom relief through treatments including steroids. Areas Covered This review summarizes safety and efficacy in promising areas of DMD therapeutics – small molecules, stop codon readthrough, gene replacement, and exon skipping – under clinical examination from 2015–2020 as demonstrated in the NIH Clinical Trials and PubMed search engines. Expert Opinion Currently, steroids persist as the most accessible medicine for DMD. Stop-codon readthrough, gene replacement, and exon-skipping therapies all aim to restore dystrophin expression. Of these strategies, gene replacement therapy has recently gained momentum while exon-skipping retains great traction. The FDA approval of three exon-skipping antisense oligonucleotides illustrate this regulatory momentum, though the effectiveness and sequence design of eteplirsen remain controversial. Cell-penetrating peptides promise to more efficaciously treat DMD-related cardiomyopathy.The recent success of antisense therapies, however, poses major regulatory challenges. To fully realize the benefits of exon-skipping, including cocktail oligonucleotide-mediated multiple exon-skipping and oligonucleotide drugs for very rare mutations, regulatory challenges need to be addressed in coordination with scientific advances.

Intraosseous transplant of dystrophin expressing chimeric (DEC) cells improves skeletal muscle function in mdx mouse model of Duchenne muscular dystrophy.

We previously reported that systemic delivery of dystrophin expressing chimeric (DEC) cells of normal (wt) and dystrophin-deficient (mdx) myoblast (MB) or mesenchymal stem cell (MSC) origin restored dystrophin expression and improved cardiac function in the mdx mouse model of Duchenne muscular dystrophy (DMD). This study evaluated the effect of intraosseous delivery of murine DEC lines of MB (MB wt /MB mdx ) and MSC (MB wt /MSC mdx ) origin on function of gastrocnemius muscle (GM). DEC lines created by ex vivo fusion were tested in the mdx mouse model of DMD: Group 1 - vehicle (control), Group 2 - non-fused 0.25 × 106 MB wt and 0.25 × 106 MSC mdx (control), Group 3 - fused 0.5 × 106 MB wt /MB mdx DEC and Group 4 - fused 0.5 × 106 MB wt /MSCmdx DEC. In situ and in vitro muscle force tests assessed GM function at 90 days post-transplant. Application of MB wt /MSC mdx and MB wt /MB mdx DEC significantly improved the fatigue ratio of GM compared to vehicle-injected controls detected by in vivo muscle force tests (0.567 ±0.116, p = 0.045 and 0.489 ±0.087, p < 0.05, respectively). MB wt /MSCmdx DEC recipients presented enhanced maximum force at tetanus (0.145 ±0.040 g/mg, p < 0.05); furthermore, recipients of MB wt /MBmdx DEC showed a significant increase in the maximum force generation rate compared to vehicle controls (4.447 ±1.090 g/s/mg, p < 0.05). The ex vivo GM force testing in MB wt /MSCmdx DEC recipients detected increased average GM force compared to vehicle and non-fused controls. Systemic-intraosseous administration of MB wt /MBmdx and MB wt /MSCmdx DEC therapy combining the myogenic and immunomodulatory properties of MB and MSC significantly improved skeletal muscle (GM) function of force and resistance to fatigue in an mdx mouse model of DMD.

Sensitive and reliable evaluation of single-cut sgRNAs to restore dystrophin by a GFP-reporter assay.

Most Duchenne muscular dystrophy (DMD) cases are caused by deletions or duplications of one or more exons that disrupt the reading frame of DMD mRNA. Restoring the reading frame allows the production of partially functional dystrophin proteins, and result in less severe symptoms. Antisense oligonucleotide mediated exon skipping has been approved for DMD, but this strategy needs repeated treatment. CRISPR/Cas9 can also restore dystrophin reading frame. Although recent in vivo studies showed the efficacy of the single-cut reframing/exon skipping strategy, methods to find the most efficient single-cut sgRNAs for a specific mutation are lacking. Here we show that the insertion/deletion (INDEL) generating efficiency and the INDEL profiles both contribute to the reading frame restoring efficiency of a single-cut sgRNA, thus assays only examining INDEL frequency are not able to find the best sgRNAs. We therefore developed a GFP-reporter assay to evaluate single-cut reframing efficiency, reporting the combined effects of both aspects. We show that the GFP-reporter assay can reliably predict the performance of sgRNAs in myoblasts. This GFP-reporter assay makes it possible to efficiently and reliably find the most efficient single-cut sgRNA for restoring dystrophin expression.

Enhanced dimethylarginine degradation improves coronary flow reserve and exercise tolerance in Duchenne muscular dystrophy carrier mice.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates postexercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac β-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in patients with DMD.NEW & NOTEWORTHY Duchenne muscular dystrophy (DMD) carriers are at risk for cardiomyopathy. The nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is released from damaged muscle in DMD and impairs exercise performance. Transgenic expression of dimethylarginine dimethylaminohydrolase to degrade ADMA prevents cardiac hypertrophy, improves cardiac function, and improves exercise tolerance in DMD carrier mice. These findings highlight the relevance of ADMA to muscular dystrophy and have important implications for therapies targeting nitric oxide in patients with DMD and DMD carriers.

Induced Fetal Human Muscle Stem Cells with High Therapeutic Potential in a Mouse Muscular Dystrophy Model.

SummaryDuchenne muscular dystrophy (DMD) is a progressive and fatal muscle-wasting disease caused by DYSTROPHIN deficiency. Cell therapy using muscle stem cells (MuSCs) is a potential cure. Here, we report a differentiation method to generate fetal MuSCs from human induced pluripotent stem cells (iPSCs) by monitoring MYF5 expression. Gene expression profiling indicated that MYF5-positive cells in the late stage of differentiation have fetal MuSC characteristics, while MYF5-positive cells in the early stage of differentiation have early myogenic progenitor characteristics. Moreover, late-stage MYF5-positive cells demonstrated good muscle regeneration potential and produced DYSTROPHIN in vivo after transplantation into DMD model mice, resulting in muscle function recovery. The engrafted cells also generated PAX7-positive MuSC-like cells under the basal lamina of DYSTROPHIN-positive fibers. These findings suggest that MYF5-positive fetal MuSCs induced in the late stage of iPSC differentiation have cell therapy potential for DMD.

Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing

CRISPR-Cas9 has emerged as a powerful technology that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) to target and edit DNA. However, many therapeutic targets cannot currently be accessed due to the lack of carriers that can deliver RNPs systemically. Here, we report a generalizable methodology that allows engineering of modified lipid nanoparticles to efficiently deliver RNPs into cells and edit tissues including muscle, brain, liver, and lungs. Intravenous injection facilitated tissue-specific, multiplexed editing of six genes in mouse lungs. High carrier potency was leveraged to create organ-specific cancer models in livers and lungs of mice though facile knockout of multiple genes. The developed carriers were also able to deliver RNPs to restore dystrophin expression in DMD mice and significantly decrease serum PCSK9 level in C57BL/6 mice. Application of this generalizable strategy will facilitate broad nanoparticle development for a variety of disease targets amenable to protein delivery and precise gene correction approaches.

Diminution in sperm quantity and quality in mouse models of Duchenne Muscular Dystrophy induced by a myostatin-based muscle growth-promoting intervention.

Duchenne Muscular Dystrophy is a devastating disease caused by the absence of a functional rod-shaped cytoplasmic protein called dystrophin. Several avenues are being developed aimed to restore dystrophin expression in boys affected by this X-linked disease. However, its complete cure is likely to need combinational approaches which may include regimes aimed at restoring muscle mass. Augmenting muscle growth through the manipulation of the Myostatin/Activin signalling axis has received much attention. However, we have recently shown that while manipulation of this axis in wild type mice using the sActRIIB ligand trap indeed results in muscle growth, it also had a detrimental impact on the testis. Here we examined the impact of administering a powerful Myostatin/Activin antagonist in two mouse models of Duchenne Muscular Dystrophy. We report that whilst the impact on muscle growth was not always positive, both models showed attenuated testis development. Sperm number, motility and ultrastructure were significantly affected by the sActRIIB treatment. Our report suggests that interventions based on Myostatin/Activin should investigate off-target effects on tissues as well as muscle.

Diminution in sperm quantity and quality in mouse models of Duchenne Muscular Dystrophy induced by a myostatin-based muscle growth-promoting intervention

Duchenne Muscular Dystrophy is a devastating disease caused by the absence of a functional rod-shaped cytoplasmic protein called dystrophin. Several avenues are being developed aimed to restore dystrophin expression in boys affected by this X-linked disease. However, its complete cure is likely to need combinational approaches which may include regimes aimed at restoring muscle mass. Augmenting muscle growth through the manipulation of the Myostatin/Activin signalling axis has received much attention. However, we have recently shown that while manipulation of this axis in wild type mice using the sActRIIB ligand trap indeed results in muscle growth, it also had a detrimental impact on the testis. Here we examined the impact of administering a powerful Myostatin/Activin antagonist in two mouse models of Duchenne Muscular Dystrophy. We report that whilst the impact on muscle growth was not always positive, both models showed attenuated testis development. Sperm number, motility and ultrastructure were significantly affected by the sActRIIB treatment. Our report suggests that interventions based on Myostatin/Activin should investigate off-target effects on tissues as well as muscle.

Diminution in sperm quantity and quality in mouse models of Duchenne Muscular Dystrophy induced by a myostatin-based muscle growth-promoting intervention

Duchenne Muscular Dystrophy is a devastating disease caused by the absence of a functional rod-shaped cytoplasmic protein called dystrophin. Several avenues are being developed aimed to restore dystrophin expression in boys affected by this X-linked disease. However, its complete cure is likely to need combinational approaches which may include regimes aimed at restoring muscle mass. Augmenting muscle growth through the manipulation of the Myostatin/Activin signalling axis has received much attention. However, we have recently shown that while manipulation of this axis in wild type mice using the sActRIIB ligand trap indeed results in muscle growth, it also had a detrimental impact on the testis. Here we examined the impact of administering a powerful Myostatin/Activin antagonist in two mouse models of Duchenne Muscular Dystrophy. We report that whilst the impact on muscle growth was not always positive, both models showed attenuated testis development. Sperm number, motility and ultrastructure were significantly affected by the sActRIIB treatment. Our report suggests that interventions based on Myostatin/Activin should investigate off-target effects on tissues as well as muscle.

Correction of Three Prominent Mutations in Mouse and Human Models of Duchenne Muscular Dystrophy by Single-Cut Genome Editing

Duchenne muscular dystrophy (DMD), one of the most common neuromuscular disorders of children, is caused by the absence of dystrophin protein in striated muscle. Deletions of exons 43, 45, and 52 represent mutational "hotspot" regions in the dystrophin gene. We created three new DMD mouse models harboring deletions of exons 43, 45, and 52 to represent common DMD mutations. To optimize CRISPR-Cas9 genome editing using the single-cut strategy, we identified single guideRNAs (sgRNAs) capable of restoring dystrophin expression by inducing exon skipping and reframing. Intramuscular delivery of AAV9 encoding SpCas9 and selected sgRNAs efficiently restored dystrophin expression in these new mouse models, offering a platform for future studies of dystrophin gene correction therapies. To validate the therapeutic potential of this approach, we identified sgRNAs capable of restoring dystrophin expression by the single-cut strategy in cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) with each of these hotspot deletion mutations. We found that the potential effectiveness of individual sgRNAs in correction of DMD mutations cannot be predicted a priori, highlighting the importance of sgRNA design and testing as a prelude for applying gene editing as a therapeutic strategy for DMD.

Engraftment of human induced pluripotent stem cell-derived myogenic progenitors restores dystrophin in mice with duchenne muscular dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is a devastating genetic muscular disorder with no effective treatment that is caused by the loss of dystrophin. Human induced pluripotent stem cells (hiPSCs) offer a promising unlimited resource for cell-based therapies of muscular dystrophy. However, their clinical applications are hindered by inefficient myogenic differentiation, and moreover, the engraftment of non-transgene hiPSC-derived myogenic progenitors has not been examined in the mdx mouse model of DMD.MethodsWe investigated the muscle regenerative potential of myogenic progenitors derived from hiPSCs in mdx mice. The hiPSCs were transfected with enhanced green fluorescent protein (EGFP) vector and defined as EGFP hiPSCs. Myogenic differentiation was performed on EGFP hiPSCs with supplementary of basic fibroblast growth factor, forskolin, 6-bromoindirubin-3′-oxime as well as horse serum. EGFP hiPSCs-derived myogenic progenitors were engrafted into mdx mice via both intramuscular and intravenous injection. The restoration of dystrophin expression, the ratio of central nuclear myofibers, and the transplanted cells-derived satellite cells were accessed after intramuscular and systemic transplantation.ResultsWe report that abundant myogenic progenitors can be generated from hiPSCs after treatment with these three small molecules, with consequent terminal differentiation giving rise to mature myotubes in vitro. Upon intramuscular or systemic transplantation into mdx mice, these myogenic progenitors engrafted and contributed to human-derived myofiber regeneration in host muscles, restored dystrophin expression, ameliorated pathological lesions, and seeded the satellite cell compartment in dystrophic muscles.ConclusionsThis study demonstrates the muscle regeneration potential of myogenic progenitors derived from hiPSCs using non-transgenic induction methods. Engraftment of hiPSC-derived myogenic progenitors could be a potential future therapeutic strategy to treat DMD in a clinical setting.

Combined Treatment with Peptide-Conjugated Phosphorodiamidate Morpholino Oligomer-PPMO and AAV-U7 Rescues the Severe DMD Phenotype in Mice

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by an absence of the dystrophin protein, which is essential for muscle fiber integrity. Among the developed therapeutic strategies for DMD, the exon-skipping approach corrects the frameshift and partially restores dystrophin expression. It could be achieved through the use of antisense sequences, such as peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) or the small nuclear RNA-U7 carried by an adeno-associated virus (AAV) vector. AAV-based gene therapy approaches have potential for use in DMD treatment but are subject to a major limitation: loss of the AAV genome, necessitating readministration of the vector, which is not currently possible, due to the immunogenicity of the capsid. The PPMO approach requires repeated administrations and results in only weak cardiac dystrophin expression. Here, we evaluated a combination of PPMO- and AAV-based therapy in a mouse model of severe DMD. Striking benefits of this combined therapy were observed in striated muscles, with marked improvements in heart and diaphragm structure and function, with unrivalled extent of survival, opening novel therapeutic perspectives for patients.

Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.

Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMDgenetic mutation in canine Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.

Effective restoration of dystrophin expression in iPSC Mdx-derived muscle progenitor cells using the CRISPR/Cas9 system and homology-directed repair technology

Duchenne muscular dystrophy (DMD) is a progressive myopathic disease caused by mutations in the gene encoding dystrophin protein that eventually leads to the exhaustion of myogenic progenitor cells (MPC). Autologous induced pluripotent stem cells (iPSCs) provide an endless source of MPC, which can potentially replenish the progenitor cell pool, repair muscle damage, and prevent DMD progression. Deletion of mutant exon 23 (ΔEx23) with clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology can correct dystrophin gene expression in iPSCs. However, successful exon23 deletion and clonal isolation are very inefficient (~3%), and manual selection of each iPSC clone and genotyping to identify ΔEx23 is labor-intensive. To overcome these obstacles, we added a homology-directed repair (HDR) donor vector, which carries floxed fluorescent protein and antibiotic selection genes, thus allowing us to identify ΔEx23 iPSC with donor selective gene integration. Our results indicate that the HDR-mediated targeted integration enables ΔEx23 iPSC identification; the HDR donor vector increased the recognition efficiency of clonal isolation (>90% as confirmed by Sanger sequencing). After removal of the inserted genes by Cre-mediated recombination followed by doxycycline (Dox)-induced MyoD induction, ΔEx23 iPSC differentiated into MPC with restored dystrophin expression in vitro. Importantly, transplanted ΔEx23 iPSC-MPC express dystrophin in the muscles of a mouse model of DMD (Mdx mice). In conclusion, the use of HDR donor vector increased the efficiency of ΔEx23 gene correction by CRISPR/Cas9, and facilitate the identification of successfully edited iPSC clones for cell therapy of DMD.

Charcot-Marie-Tooth type 4B2 demyelinating neuropathy in miniature Schnauzer dogs caused by a novel splicing SBF2 (MTMR13) genetic variant: a new spontaneous clinical model.

BackgroundCharcot-Marie-Tooth (CMT) disease is the most common neuromuscular disorder in humans affecting 40 out of 100,000 individuals. In 2008, we described the clinical, electrophysiological and pathological findings of a demyelinating motor and sensory neuropathy in Miniature Schnauzer dogs, with a suspected autosomal recessive mode of inheritance based on pedigree analysis. The discovery of additional cases has followed this work and led to a genome-wide association mapping approach to search for the underlying genetic cause of the disease.MethodsFor genome wide association screening, genomic DNA samples from affected and unaffected dogs were genotyped using the Illumina CanineHD SNP genotyping array. SBF2 and its variant were sequenced using primers and PCRs. RNA was extracted from muscle of an unaffected and an affected dog and RT-PCR performed. Immunohistochemistry for myelin basic protein was performed on peripheral nerve section specimens.ResultsThe genome-wide association study gave an indicative signal on canine chromosome 21. Although the signal was not of genome-wide significance due to the small number of cases, the SBF2 (also known as MTMR13) gene within the region of shared case homozygosity was a strong positional candidate, as 22 genetic variants in the gene have been associated with demyelinating forms of Charcot-Marie-Tooth disease in humans. Sequencing of SBF2 in cases revealed a splice donor site genetic variant, resulting in cryptic splicing and predicted early termination of the protein based on RNA sequencing results.ConclusionsThis study reports the first genetic variant in Miniature Schnauzer dogs responsible for the occurrence of a demyelinating peripheral neuropathy with abnormally folded myelin. This discovery establishes a genotype/phenotype correlation in affected Miniature Schnauzers that can be used for the diagnosis of these dogs. It further supports the dog as a natural model of a human disease; in this instance, Charcot-Marie-Tooth disease. It opens avenues to search the biological mechanisms responsible for the disease and to test new therapies in a non-rodent large animal model. In particular, recent gene editing methods that led to the restoration of dystrophin expression in a canine model of muscular dystrophy could be applied to other canine models such as this before translation to humans.

Characterization of mesoangioblast cell fate and improved promyogenic potential of a satellite cell-like subpopulation upon transplantation in dystrophic murine muscles.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease caused by the lack of dystrophin in muscle fibersthat is currently without curative treatment. Mesoangioblasts (MABs) are multipotent progenitor cells that can differentiate to a myogenic lineage and that can be used to express Dystrophin upon transplantation into muscles, in autologous gene therapy approaches. However, their fate in the muscle environment remains poorly characterized. Here, we investigated the differentiation fate of MABs following their transplantation in DMD murine muscles using a mass cytometry strategy. This allowed the identification and isolation of a fraction of MAB-derived cells presenting common properties with satellite muscle stem cells. This analysis also indicated that most cells did not undergo a myogenic differentiation path once in the muscle environment, limiting their capacity to restore dystrophin expression in transplanted muscles. We therefore assessed whether MAB treatment with cytokines and growth factors prior to engraftment may improve their myogenic fate. We identified a combination of such signals that ameliorates MABs capacity to undergo myogenic differentiation in vivo and to restore dystrophin expression upon engraftment in myopathic murine muscles.

P.265Integrating clinical outcome measures, MRI and circulating biomarkers in subjects with Becker muscular dystrophy: a prospective longitudinal study

Becker muscular dystrophy (BMD) is caused by mutations in the dystrophin (DMD) gene. BMD is clinically characterized by a highly variable disease course ranging from mild symptoms to severe muscle weakness. Natural history studies in BMD subjects are required to prepare for clinical trials in this population as well as to understand what possible outcomes may be expected in pharmacological interventions aimed at restoring dystrophin expression in subjects affected by Duchenne MD. Until now, most studies have focused on a single or limited set of biomarkers. In the present study, we will present an approach that combines a large number of potential biomarkers to improve prognostic prediction in BMD. In this prospective study we obtained longitudinal and cross-sectional clinical, muscle MRI and serum biomarker data from 52 genetically confirmed BMD subjects with a follow-up of 2 to 8 years. Clinical tests included quantitative muscle assessment of the major muscle groups, the 6-Minute Walk Distance (6-MWD) test, Performance of the Upper Limb (PUL) and the North Star Ambulatory Assessment (NSAA). In a subgroup of 23 subjects, 3-point Dixon MRI scans were acquired and used to calculate weighted average fat fraction and contractile cross-sectional area of the middle of the quadriceps muscle. Serum samples were collected yearly to quantify a panel of biomarkers linked to the pathophysiology such as TGF-β factors, to the metabolic state of muscle such as creatine and creatinine, or to the mutation site (i.e. mutation affecting or not affecting the NOS binding site). Association and integration of the different readouts will be presented including testing on how MRI, clinical and circulating biomarker data can be integrated to improve the prognostic prediction of disease progression in BMD patients. Comparison with DMD patients will also be presented.

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.