Therapy For Duchenne Muscular Dystrophy Research Articles

Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of the protein dystrophin. Utrophin is a dystrophin homologue currently under investigation as a protein replacement therapy for Duchenne muscular dystrophy. Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma. While utrophin is homologous with dystrophin from a molecular and biochemical perspective, we have recently shown that full-length utrophin expressed in eukaryotic cells is stiffer than what has been reported for dystrophin fragments expressed in bacteria. In this study, we show that differences in expression system impact the mechanical stiffness of a model utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3). We also demonstrate that UtrN-R3 expressed in eukaryotic cells was phosphorylated while bacterial UtrN-R3 was not detectably phosphorylated. Using atomic force microscopy, we show that phosphorylated UtrN-R3 exhibited significantly higher unfolding forces compared to unphosphorylated UtrN-R3 without altering its actin-binding activity. Consistent with the effect of phosphorylation on mechanical stiffness, mutating the phosphorylated serine residues on insect eukaryotic protein to alanine decreased its stiffness to levels not different from unphosphorylated bacterial protein. Taken together, our data suggest that the mechanical properties of utrophin may be tuned by phosphorylation, with the potential to improve its efficacy as a protein replacement therapy for dystrophinopathies.

Diverse effector and regulatory functions of fibro/adipogenic progenitors during skeletal muscle fibrosis in muscular dystrophy

Fibrosis is a prominent pathological feature of skeletal muscle in Duchenne muscular dystrophy (DMD). The commonly used disease mouse model, mdx 5cv , displays progressive fibrosis in the diaphragm but not limb muscles. We use single-cell RNA sequencing to determine the cellular expression of the genes involved in extracellular matrix (ECM) production and degradation in the mdx 5cv diaphragm and quadriceps. We find that fibro/adipogenic progenitors (FAPs) are not only the primary source of ECM but also the predominant cells that express important ECM regulatory genes, including Ccn2, Ltbp4, Mmp2, Mmp14, Timp1, Timp2, and Loxs. The effector and regulatory functions are exerted by diverse FAP clusters which are different between diaphragm and quadriceps, indicating their activation by different tissue microenvironments. FAPs are more abundant in diaphragm than in quadriceps. Our findings suggest that the development of anti-fibrotic therapy for DMD should target not only the ECM production but also the pro-fibrogenic regulatory functions of FAPs.

PCR51 Reassessing the Acceptability of Risk of Death Associated With Gene Therapy for Duchenne Muscular Dystrophy: A Qualitative Study to Inform Updating a Quantitative Instrument

In 2017/2018, we concluded a study on risk tolerance for gene therapy (GT) for Duchenne muscular dystrophy (Duchenne). Duchenne is a progressive, fatal condition. GTs under trial could be disease modifying, not curative. GT has serious risks, including risk of death. In our prior study we assessed maximum acceptable risk (MAR) of death in U.S.-based adults with Duchenne and parents. In GT trials over the past 5 years there were serious adverse events, clinical holds, and one death in late 2021. We initiated a second study to assess current MAR in the U.S., United Kingdom, and to explore experiences and attitudes of clinicians.

PCR31 Patient and Caregiver Preferences for the Treatment of Duchenne Muscular Dystrophy: A Quantitative Patient Preference Study

With gene replacement therapy for Duchenne muscular dystrophy (DMD) being evaluated in phase 3 clinical trials, a crucial step is made to address numerous unmet needs of these patients. Since this therapy is associated with uncertainties regarding the benefits, side effects, and risks, this research wants to quantify the patient treatment needs and their acceptability towards treatment alternatives. The final objective is to inform regulatory, HTA/payer (reimbursement), and clinical practice decision making.

Use of Glycine to Augment Exon Skipping and Cell Therapies for Duchenne Muscular Dystrophy.

Antisense oligonucleotide (AO)-based exon-skipping and cell therapies are the main therapeutic approaches for Duchenne muscular dystrophy (DMD). Insufficient systemic delivery leading to low therapeutic efficacy limits the former; low transplantation efficiency hampers the latter. Here we describe how glycine can address these issues by augmenting satellite proliferation and muscle regeneration, resulting in enhanced AO uptake in regenerating myofibers and cell transplantation efficiency in dystrophic mice. The dual functionality of glycine demonstrated in AO-based exon-skipping and cell therapies presents a simple and efficient method to augment AO potency and cell transplantation efficacy in DMD and other muscle diseases.

A state-of-the-art review of tamoxifen as a potential therapeutic for duchenne muscular dystrophy.

Introduction: This systematic review analyzes the state-of-art repurposing of the drug tamoxifen (TAM) in the treatment of Duchenne Muscular Dystrophy (DMD), including its mechanism of action, toxicological findings, and past and ongoing clinical trials. A parallel aim of this work was to explore whether evidence exists to support further funding of investigation on TAM treatment for DMD patients with a pivotal trial in young patients. Bringing evidence and answering the scientific question of whether this treatment could improve the quality-of-life of DMD patients is needed to establish guidelines and accelerate access to promising therapies for DMD patients. Methods: The search was conducted in January 2022 utilizing PubMed. All MeSH terms for "Duchenne Muscular Dystrophy" and "tamoxifen" were used. The inclusion and exclusion criteria were defined according to the PICOS framework. Results: The included publications all explored the use of TAM with promising outcomes in muscular strength recovery and a decrease in pathology biomarkers. Two reviews recognize TAM as a potential treatment for DMD patients and state that drug repurposing plays a crucial role in the quest for a drug candidate to treat this rare disease. Conclusion: According to available data, TAM shows promise as a treatment for DMD, both pharmacologically and clinically. However, published data to date are insufficient to definitively conclude the beneficial effect of TAM on quality-of-life and ultimately survival, particularly in the youngest patients diagnosed with DMD.

Enhancing interaction of actin and actin-binding domain 1 of dystrophin with modulators: Toward improved gene therapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full- length dystrophin. Thus increasing the actin-binding affinity of micro-dystrophin, using small molecules, could be a beneficial therapeutic approach. Here we have developed and validated a novel high-throughput screening (HTS) assay to discover small molecules that increase the binding affinity of dystrophin's actin-binding domain1 (ABD1). We engineered a novel fluorescence resonance energy transfer (FRET) biosensor, consisting of the mClover3, fluorescent protein (donor) attached to the C- terminus of dystrophin ABD1, and Alexa Fluor 568 (acceptor) attached to the C-terminal cysteine of actin. We used this biosensor in small-molecule screening, using a unique high-precision, high-throughput screening (HTS) fluorescence lifetime assay, identifying several compounds from a FDA-approved library that significantly increase the binding between actin and ABD1. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the actin-dystrophin interaction, with the ultimate goal of developing therapies for muscular dystrophy.

Gene Therapy for Duchenne Muscular Dystrophy: Unlocking the Opportunities in Countries in the Middle East and Beyond.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder which leads to progressive muscle degeneration and weakness. Most patients die from cardiac or respiratory failure. Gene transfer therapy offers a promising approach to treating this disorder. Given the genetic disease burden, family size, and the high consanguinity rates in the Middle East, our objective is to address current practices and challenges of DMD patient care within two countries in this region, namely the United Arab Emirates and Kuwait, and to outline readiness for gene therapy. An expert panel meeting was held to discuss the DMD patient journey, disease awareness, current management of DMD, challenges faced and recommendations for improvement. Opportunities and challenges for gene therapy in both countries were also deliberated. A pre-meeting survey was conducted, and the results were used to guide the discussion during the meeting. DMD awareness is poor resulting in a delay in referral and diagnosis of patients. Awareness and education initiatives, along with an interconnected referral system could improve early diagnosis. Genetic testing is available in both countries although coverage varies. Corticosteroid therapy is the standard of care however there is often a delay in treatment initiation. Patients with DMD should be diagnosed and managed by a multi-disciplinary team in centers of excellence for neuromuscular disorders. Key success factors to support the introduction of gene therapy include education and training, timely and accessible genetic testing and resolution of reimbursement and cost issues. There are many challenges facing the management of DMD patients in the United Arab Emirates and Kuwait and most likely other countries within the Middle East. Successful introduction of gene therapy to treat DMD will require careful planning, education, capacity building and prioritization of core initiatives.

CRISPR-mediated correction of skeletal muscle Ca2+ handling in a novel DMD patient-derived pluripotent stem cell model

Mutations in the dystrophin gene cause the most common and currently incurable Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting. Although abnormal Ca2+ handling is a pathological feature of DMD, mechanisms underlying defective Ca2+ homeostasis remain unclear. Here we generate a novel DMD patient-derived pluripotent stem cell (PSC) model of skeletal muscle with an isogenic control using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated precise gene correction. Transcriptome analysis identifies dysregulated gene sets in the absence of dystrophin, including genes involved in Ca2+ handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca2+ transients and mathematical modeling of Ca2+ dynamics reveal significantly reduced cytosolic Ca2+ clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca2+ flux in myotubes is determined by both intracellular and extracellular sources. DMD-PSC derived myotubes display significantly reduced velocity of contractility. Compared with a non-isogenic wildtype PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca2+ homeostasis in DMD and suggests that Ca2+ signaling pathways amenable to pharmacological modulation are potential therapeutic targets. Importantly, we have established a human physiology-relevant in vitro model enabling rapid pre-clinical testing of potential therapies for DMD.

A microtubule-connexin-43 regulatory link suppresses arrhythmias and cardiac fibrosis in Duchenne muscular dystrophy mice.

Dilated cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD), an inherited degenerative disease of the cardiac and skeletal muscle caused by absence of the protein dystrophin. We showed one hallmark of DMD cardiomyopathy is the dysregulation of cardiac gap junction channel protein connexin-43 (Cx43). Proper Cx43 localization and function at the cardiac intercalated disc (ID) is regulated by post-translational phosphorylation of Cx43-carboxy-terminus residues S325/S328/S330 (pS-Cx43). Concurrently, Cx43 traffics along microtubules (MTs) for targeted delivery to the ID. In DMD hearts, absence of dystrophin results in a hyperdensified and disorganized MT cytoskeleton, yet the link with pS-Cx43 remains unaddressed. To gain insight into the relationship between MTs and pS-Cx43, DMD mice (mdx) and pS-Cx43-deficient (mdxS3A) mice were treated with an inhibitor of MT polymerization, colchicine (Colch). Colch treatment protected mdx, not mdxS3A mice, against Cx43 remodeling, improved MT directionality, and enhanced pS-Cx43/tubulin interaction. Likewise, severe arrhythmias were prevented in isoproterenol-stressed mdx, not mdxS3A mice. Furthermore, MT directionality was improved in pS-Cx43-mimicking mdx (mdxS3E). Mdxutr+/- and mdxutr+/-S3A mice, lacking one copy of dystrophin homolog utrophin, displayed enhanced cardiac fibrosis and reduced lifespan compared with mdxutr+/-S3E; and Colch treatment corrected cardiac fibrosis in mdxutr+/- but not mdxutr+/-S3A. Collectively, the data suggest that improved MT directionality reduces Cx43 remodeling and that pS-Cx43 is necessary and sufficient to regulate MT organization, which plays crucial role in correcting cardiac dysfunction in DMD mice. Thus, identification of novel organizational mechanisms acting on pS-Cx43-MT will help develop novel cardioprotective therapies for DMD cardiomyopathy.NEW & NOTEWORTHY We found that colchicine administration to Cx43-phospho-deficient dystrophic mice fails to protect against Cx43 remodeling. Conversely, Cx43-phospho-mimic dystrophic mice display a normalized MT network. We envision a bidirectional regulation whereby correction of the dystrophic MTs leads to correction of Cx43 remodeling, which in turn leads to further correction of the MTs. Our findings suggest a link between phospho-Cx43 and MTs that provides strong foundations for novel therapeutics in DMD cardiomyopathy.

Development of Cell Therapy for Duchenne Muscular Dystrophy by iPS Cell-derived Muscle Stem Cell, and Potential of Regenerative Rehabilitation with Cell Therapy

デュシェンヌ型筋ジストロフィーはジストロフィン欠損による筋損傷により重度の筋力低下をきたす遺伝性筋疾患である．その治療法として，骨格筋幹細胞移植による筋再生医療が期待されている．われわれは発生段階を模倣した分化誘導法の至適化により，iPS細胞から効率よく骨格筋幹細胞を誘導することに成功し，移植によりマウスモデルで高い筋再生能をもつことも示された．さらには運動機能の評価方法として足関節底屈トルク測定機器を用いた手法を開発し，約10％以上のジストロフィン発現回復と機能回復が相関する可能性を見いだしている．また，同機器によってリハビリテーション介入も可能になったことから，細胞移植治療に有用な運動プロトコルも開発した．

Gene editing of Duchenne muscular dystrophy using biomineralization-based spCas9 variant nanoparticles

The CRISPR/Cas9 mediated genome editing have provided a promising strategy to correct multiple mutations of Duchenne muscular dystrophy (DMD). However, the delivery of CRISPR/Cas9 system into mammalian cell for DMD gene editing mainly relies on adeno associated virus (AAV)-mediated transport. Meanwhile, the protospacer adjacent motif (PAM) requirement of wild-typed Cas9 protein causing the target sites for exon splice acceptor site are restricted to limited regions. Here, we developed a biomineralized PAMLess Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo. This method described a facile synthesis of biomineralized NPs with high SpRY pDNA encapsulation efficiency. In vitro results show that the Bm-SpRY NPs have the obvious advantages of well biocompatibility and protecting SpRY pDNA from enzyme degradation and efficient delivery under high serum condition. Cell studies demonstrated that Bm-SpRY NPs enable rapid cellular uptake, endo-lysosomes escape and nucleus transport. Meanwhiles, the DMD gene editing via Bm-SpRY NPs pathway is transient process without genomic integration. We evaluated multiple target regions with different PAMs for the DMD exon 51 splice acceptor site through Bm-SpRY NPs method and found that the target region with TAG PAM has the highest editing efficiency and significant preferential mutation. In vivo results show that intramuscular injection of Bm-SpRY NPs enable DMD gene mutation in muscle tissue without tissue damage. This study may extend the advanced application of CRISPR system for DMD therapy. Statement of significanceThe gene editing technology of CRISPR/Cas9 provides an effective treatment strategy for the Duchenne muscular dystrophy (DMD) therapy. However, the delivery of CRISPR system in mammalian cell mainly relies on viral mediated transport and the NGG or NAG requirement of wild-typed Cas9 protein limits the target region in DMD gene. Here, the present study provides a biomineralized PAM Less Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo. This study may extend the application of CRISPR system for DMD gene therapy.

Pharmacological TRPC6 inhibition improves survival and muscle function in mice with Duchenne muscular dystrophy.

Gene mutations causing loss of dystrophin result in the severe muscle disease known as Duchenne muscular dystrophy (DMD). Despite efforts at genetic repair, DMD therapy remains largely palliative. Loss of dystrophin destabilizes the sarcolemmal membrane, inducing mechanosensitive cation channels to increase calcium entry and promote cell damage and, eventually, muscle dysfunction. One putative channel is transient receptor potential canonical 6 (TRPC6); we have shown that TRPC6 contributed to abnormal force and calcium stress-responses in cardiomyocytes from mice lacking dystrophin that were haplodeficient for utrophin (mdx/utrn+/- [HET] mice). Here, we show in both the HET mouse and the far more severe homozygous mdx/utrn-/- mouse that TRPC6 gene deletion or its selective pharmacologic inhibition (by BI 749327) prolonged survival 2- to 3-fold, improving skeletal and cardiac muscle and bone defects. Gene pathways reduced by BI 749327 treatment most prominently regulated fat metabolism and TGF-β1 signaling. These results support the testing of TRPC6 inhibitors in human trials for other diseases as a novel DMD therapy.

VP.82 PK/PD modelling to inform clinical development of an adeno-associated virus gene transfer therapy for Duchenne muscular dystrophy

Delandistrogene moxeparvovec (SRP-9001) is an investigational gene transfer therapy for Duchenne muscular dystrophy (DMD) developed for targeted skeletal and cardiac muscle expression of micro-dystrophin—a shortened, functional dystrophin protein. We evaluated vector biodistribution, expression, and clinical dose selection of delandistrogene moxeparvovec using a novel application of a pharmacokinetic (PK) and pharmacodynamic (PD) modelling approach applied to data collected from an animal model of DMD (DMD^MDX mice). We analysed PK/PD relationships between dose, tissue vector genome exposure, micro-dystrophin protein expression (percentage dystrophin positive fibres [PDPF] and western blot), and functional improvement (relative specific force from tibialis anterior and diaphragm). Linear kinetics with a dose-proportional increase in tissue drug exposure was demonstrated across the nearly 10-fold dose range (4.43x10¹³–4.01x10¹⁴ vg/kg), and in all tissues. The relationship between tissue vector exposure and PD endpoints (PDPF, motor function outcome) showed a saturable response across a wide range of vector exposures. The vector exposure at 1.33x10¹⁴ vg/kg (the clinical dose) approached the maximum treatment response. Relative specific force and PDPF were significantly correlated (P=4.43x10^-6). However, the relationship appeared to be nonlinear, with increased PDPF expression approaching the maximal functional improvement. Relative specific force and western blot were not significantly correlated. For the first time, biodistribution, biomarker, and functional efficacy data were used to quantify and demonstrate PK/PD relationships for an adeno-associated virus (AAV)-based gene transfer therapy in a DMD animal model. The results continue to support the expected therapeutic benefit and clinical dose of delandistrogene moxeparvovec, an AAV-based gene transfer therapy.

Newborn Screening for Duchenne Muscular Dystrophy: First Year Results of a Population-Based Pilot.

Advancements in therapies for Duchenne muscular dystrophy (DMD) have made diagnosis within the newborn period a high priority. We undertook a consortia approach to advance DMD newborn screening in the United States. This manuscript describes the formation of the Duchenne Newborn Screening Consortium, the development of the pilot protocols, data collection tools including parent surveys, and findings from the first year of a two-year pilot. The DMD pilot design is population-based recruitment of infants born in New York State. Data tools were developed to document the analytical and clinical validity of DMD NBS, capture parental attitudes, and collect longitudinal health information for diagnosed newborns. Data visualizations were updated monthly to inform the consortium on enrollment. After 12 months, 15,754 newborns were screened for DMD by the New York State Newborn Screening (NYS NBS) Program. One hundred and forty screened infants had borderline screening results, and sixteen infants were referred for molecular testing. Three male infants were diagnosed with dystrophinopathy. Data from the first year of a two-year NBS pilot for DMD demonstrate the feasibility of NBS for DMD. The consortia approach was found to be a useful model, and the Newborn Screening Translational Research Network’s data tools played a key role in describing the NBS pilot findings and engaging stakeholders.

Exon skipping induces uniform dystrophin rescue with dose-dependent restoration of serum miRNA biomarkers and muscle biophysical properties

Therapies that restore dystrophin expression are presumed to correct Duchenne muscular dystrophy (DMD), with antisense-mediated exon skipping being the leading approach. Here we aimed to determine whether exon skipping using a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugate results in dose-dependent restoration of uniform dystrophin localization, together with correction of putative DMD serum and muscle biomarkers. Dystrophin-deficient mdx mice were treated with a PPMO (Pip9b2-PMO) designed to induce Dmd exon 23 skipping at single, ascending intravenous doses (3, 6, or 12 mg/kg) and sacrificed 2 weeks later. Dose-dependent exon skipping and dystrophin protein restoration were observed, with dystrophin uniformly distributed at the sarcolemma of corrected myofibers at all doses. Serum microRNA biomarkers (i.e., miR-1a-3p, miR-133a-3p, miR-206-3p, miR-483-3p) and creatinine kinase levels were restored toward wild-type levels after treatment in a dose-dependent manner. All biomarkers were strongly anti-correlated with both exon skipping level and dystrophin expression. Dystrophin rescue was also strongly positively correlated with muscle stiffness (i.e., Young’s modulus) as determined by atomic force microscopy (AFM) nanoindentation assay. These data demonstrate that PPMO-mediated exon skipping generates myofibers with uniform dystrophin expression and that both serum microRNA biomarkers and muscle AFM have potential utility as pharmacodynamic biomarkers of dystrophin restoration therapy in DMD.

Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1-targeted delivery of antisense oligonucleotide using FORCE conjugation in mdx mice.

Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE-M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE-M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90%and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE-M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.

Abstract P2107: Improving Stem Cell Models Of Duchenne Muscular Dystrophy-related Cardiomyopathy By Incorporation Of Physiologic Mechanical Stress

Background: Cardiomyopathy in Duchenne muscular dystrophy (DMD) is a significant driver of disease morbidity and mortality. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer the opportunity to study patient-specific mutations in the dish, however they generally suffer from immaturity. DMD is caused by mutations in the gene DMD , which codes for the protein dystrophin. Dystrophin localizes to the sarcolemma, forming part of a critical transmembrane mechanical and signaling complex that bridges the sarcomere to the extracellular matrix. Disruption of this axis leads to membrane fragility and mechanical stress is a main driver of DMD pathology. New therapies for DMD are in clinical development, including exon skipping, micro-dystrophin gene therapy, CRISPR-based gene editing, and membrane resealants, with some exon skipping strategies receiving FDA approval. The main outcomes of these therapies have been tied to improvement in skeletal muscle function with cardiac efficacy remaining understudied, despite its clear importance for patient outcomes. Results: Equibiaxial strain was employed as a physiologic mechanical stressor to simulate the in vivo mechanism of myocyte injury in an in vitro format using patient-derived DMD iPSC-CMs. By titrating strain, we determined that application of 10% strain for 2 hours robustly differentiates DMD and control iPSC-CMs using the clinically relevant biomarker LDH release as a proxy for membrane damage (control LDH release fold change: no significant change; DMD LDH release fold change: 2.51, p<0.0001). We evaluated DMD biomarkers released into the media using aptamer-based proteomics profiling and identified a striking strong correlation with serum biomarkers from DMD patients. Longer duration strain (24 hours) led to cell injury in both control and DMD iPSC-CMs. A membrane resealant was evaluated in this platform, which demonstrated enhanced recovery following treatment for both DMD and control iPSC-CMs with concomitant reduction in biomarker release. Conclusions: iPSC-CM models can be improved by incorporating physiologic mechanical stress, which is a main driver pathology. This platform is especially useful for evaluating dystrophic cardiomyopathy therapeutics.

The new challenge of "exercise + X″ therapy for Duchenne muscular dystrophy-Individualized identification of exercise tolerance and precise implementation of exercise intervention.

Duchenne muscular dystrophy (DMD) is an X-linked recessive fatal muscular disease. Gene therapy, cell therapy, and drug therapy are currently the most widely used treatments for DMD. However, many experiments on animals and humans suggested that appropriate exercise could improve the effectiveness of such precision medicine treatment, thereby improving patient’s muscle quality and function. Due to the striated muscle damage of DMD individuals, there are still many debates about whether DMD animals or patients can exercise, how to exercise, when to exercise best, and how to exercise effectively. The purpose of this review is to summarize and investigate the scientific basis and efficacy of exercise as an adjuvant therapy for DMD gene therapy, cell therapy and drug therapy, as well as to present the theoretical framework and optional strategies of “exercise + X″″ combination therapy.

Abstract P2069: In Vivo Investigation Of The Mechanisms Regulating Truncated Dystrophin Protein Turnover In The Heart

Numerous inherited and acquired cardiac diseases are directly linked to a deficiency in the protein dystrophin, such as the development of severe cardiomyopathy in Duchenne Muscular Dystrophy (DMD) patients. Dilated cardiomyopathy, arrhythmias, and congestive heart failure represent the most important life-limiting condition in DMD. Currently, there is great excitement in the muscular dystrophy community as multiple ongoing gene-based therapies, are advancing through clinical trials. These therapies utilize miniaturized dystrophin constructs, and these modified dystrophins are the focus of this study. We implemented an innovative and valuable system to determine the direct physiological significance of dystrophin protein deficiency including determining the half-life of truncated dystrophin constructs. This system provides a novel means to investigate mechanistically how deletions in dystrophin affect therapeutically shortened dystrophin turnover in cardiac muscle in vivo. We used a floxed allele approach together with a cardiac directed (αMHC Mer-Cre-Mer) inducible Cre for precise control of full-length dystrophin or therapeutic micro-dystrophin gene excision. We examined the time course of full-length and micro-dystrophin mRNA as a biologically relevant surrogate for intact gene excision efficiency. Our data showed evidence of significant full-length dystrophin cDNA and micro-dystrophin gene excision, with a complete loss of micro-dystrophin mRNA and a gene excision efficiency of 80% for full-length dystrophin. Heart tissues were then extracted for protein quantitative analysis of micro-dystrophin (5, 10 and 30 days) and of full-length dystrophin (1, 3 and 6 months) post tamoxifen administration. Results reported a fast turnover rate for micro-dystrophin in the heart, with calculated half-life of between 5-7 days. In marked contrast, full-length dystrophin was highly stable with dystrophin protein content (~ 40%) at 6 months, after dystrophin gene excision. Studies of in vivo full-length and micro-dystrophin protein half-life in skeletal muscle, are ongoing. This work will provide key information required for long-term success of ongoing and future DMD therapies featuring gene therapy with shortened dystrophins.