Mutations In Dystrophin Gene Research Articles

571. Precise Control of CRISPR-Cas9 Mediated Gene Editing for Correcting Mutation of Duchenne Muscular Dystrophy in iPS Cells

Duchenne Muscular Dystrophy (DMD) is one of the most common and severe inherited neuromuscular disease caused by the loss-of-function mutations in Dystrophin gene on X chromosome. Currently, no effective treatment available for DMD, including gene augmentation therapy, since large size of the Dystrophin gene hamper the delivery by viral vectors. Exon skipping to modulate mRNA splicing patterns using antisense oligonucleotide is a promising approach currently investigated in clinical trials, however, the effect of antisense oligos is transient.Recent progress on targeted gene editing by engineered nucleases, such as TALENs or CRISPR/Cas9, have evolutionally broaden our ability to precisely modify the genomic sequence at desired locus, including genetic correction of Dystrophin gene. At the same time, however, undesired mutagenesis at the off-target loci with a few base-pair mismatches is a big concern.To this end, we previously developed a method to identify “unique sequence” in the human genome by using a database of short and unique k-mer sequences [Li HL et al., Stem Cell Reports, 4(1):143-54, 2015]. Our “uniqueness” data is useful to design a target site for engineered nucleases with high sequence specificity and minimum number of putative off-target sites, and the data is publically available at https://apps.cira.kyoto-u.ac.jp/igeats/.Moreover, to precisely control the genomic cleavage activity of CRISPR-Cas9, we constructed doxycycline inducible expression vector and Cas9 protein fused a nuclear receptor to mediate nuclear shuttling upon addition of receptor substrate. We show that both transcriptional and posttranscriptional regulations of CRISPR-Cas9 protein are effective to modulate DNA cleavage activity.To test the effectiveness of CRISPR system to correct disease mutation, we took advantages of patient-derived induced pluripotent stem cells (iPSCs) as a platform for testing various gene correction approaches. As a disease modeling for DMD gene thrapy, we derived integration-free iPS cell lines from a DMD patient who lacks the exon 44 in the Dystrophin gene, which is one of the most comon deletions of single exon. We will evaluate the dual-controlable Cas9 approache in patient-derived iPSCs, since gene-corrected iPSCs hold a promise to serve as a cell source for future DMD therapy.

148. Screening and Optimization of Antisense Oligonucleotide for Skipping Human Dystrophin Exon 51 and 53

Duchenne muscular dystrophy (DMD) results from dystrophin gene mutations, causing shift of the reading frame and preventing production of a functional protein. Most DMD mutations occur in the parts of the gene that are not critical for its function, therefore restoration of the reading frame by antisense oligonucleotide-mediated exon skipping is a viable approach. The efficacy of antisense therapy has now been proven in animal models and in clinical trials.Therapeutic effect of exon skipping largely depends on the efficiency of individual antisense oligoneotide, which has to be identified by screening. This study aims specifically to search for the most effective morpholino (PMO) oligomer to target the human dystrophin exon 51 and exon 53 for the correction of the relevant DMD mutations. We established the GFP reporter myoblast cell cultures and screened more than 40 PMOs targeting each of exon 51 and exon 53. We also examined the PMOs in normal human myoblast cultures and in humanized DMD (hDMD) mice with local delivery by i.m injection to identify PMOs of maximal skipping potency. Finally we selected 5 oligomers as vivo-PMOs targeting each of the exon 51 and 53 and examined their exon skipping efficiency in the hDMD mice by systemic delivery. We were able to identify PMOs with high exon skipping efficiency in all muscles. The selected PMOs will be further validated in patient-derived fibroblasts and then be applied to clinical trials for DMD treatment.

P167 – 2623: The wide spectrum of neuropsychiatric profile in DMD in relation to underlying dystrophin gene mutations

Objective Duchenne muscular dystrophy (DMD) is associated with neurodevelopmental disorders. The aim of the study was to fully characterize the neuropsychiatric profile and to explore underlying genotype/phenotype associations with brainexpressed dystrophin isoforms. Methods 130 DMD boys (mean age = 9.5 years) in four European neuromuscular centres (i.e. London, Brussels, Rome and Messina) were included in the study and completed IQ assessment and a screening questionnaire. Of these, 87 underwent complete neurodevelopmental assessment with structured diagnostic interview and parent reported questionnaire. The results were correlated to the underlying dystrophin gene mutations and the deduced pattern of brain dystrophin isoform expression. Results The overall mean score on the screening neurodevelopmental questionnaire was significantly abnormal compared to the general paediatric population (p Conclusion Boys with DMD are at very high risk of neuropsychiatric disturbance, including clinically severe difficulties with intellectual development, social-communication, flexibility, anxiety, hyperactivity and attention. This risk increases with mutations at the 3' end of the gene. Patterns of symptom clusters suggest a DMD neuropsychiatric syndrome, which require prompt evaluation and early intervention. Our findings further emphasize the role that dystrophin plays in brain development and function.

Clinical and Genetic Characterization of Female Dystrophinopathy

Background and PurposeDuchenne and Becker muscular dystrophies are the most common X-linked recessive muscular dystrophies. Dystrophin gene mutations usually affect men, but reportedly 2.5-7.8% of women are affected and are classified as symptomatic carriers. The aim of this study was to clinically and genetically characterize symptomatic female dystrophinopathy carriers.MethodsThe clinical and genetic data of 11 female dystrophinopathy carriers among 285 patients who underwent multiplex ligation-dependent probe amplification (MLPA) analysis for the dystrophin gene were reviewed. Women with muscle weakness and/or dilated cardiomyopathy were classified as symptomatic carriers, while subjects with high serum creatine kinase (CK) levels and/or minor myopathic signs such as muscle cramps and myalgia were classified as asymptomatic.ResultsTwelve female carriers were identified, but 1 symptomatic carrier who also had Turner syndrome was excluded from the study. Of the 11 included female carriers, 4 were symptomatic and 7 were asymptomatic. The age at symptom onset in the symptomatic female carriers ranged from 15 to 31 years (mean, 30.6 years), and the age at diagnosis for asymptomatic carriers ranged from 4 to 38 years (mean, 24.5 years). Serum CK levels were markedly elevated (mean, 1,301 IU/mL) in three of the four (75%) symptomatic female carriers, and mildly elevated in three of the seven (42%) asymptomatic female carriers. Symptomatic female carriers typically presented with asymmetric bilateral leg weakness as the initial symptom, with aggravated symptoms after labor.ConclusionsFemale dystrophinopathy is not uncommon, and it is an important factor with respect to males with dystrophinopathy who may be born to such patients. Screening with MLPA is useful because it can aid in early diagnosis and appropriate management.

The immune system in Duchenne muscular dystrophy: Friend or foe.

Duchenne muscular dystrophy (DMD) is a genetic disease caused by mutations in the X-linked dystrophin gene, resulting in reduced or absent protein production, subsequently leading to the structural instability of the dystroglycan complex (DGC), muscle degeneration, and early death in males. Thus, current treatments have been targeting the genetic defect either by bypassing the mutation through exon skipping or replacing the defective gene through gene therapy and stem cell approaches. However, what has been an underappreciated mediator of muscle pathology and, ultimately, of muscle degeneration and fibrotic replacement, is the prominent inflammatory response. Of potentially critical importance, however, is the fact that the elements mediating the inflammatory response also play an essential role in tissue repair. In this opinion piece, we highlight the detrimental and supportive immune parameters that occur as a consequence of the genetic disorder and discuss how changes to immunity can potentially ameliorate the disease intensity and be employed in conjunction with efforts to correct the genetic disorder.

Intellectual ability in the duchenne muscular dystrophy and dystrophin gene mutation location.

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy during childhood. Mutations in dystrophin (DMD) gene are also recognized as a cause of cognitive impairment. We aimed to determine the association between intelligence level and mutation location in DMD genes in Serbian patients with DMD. Forty-one male patients with DMD, aged 3 to 16 years, were recruited at the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia. All patients had defined DMD gene deletions or duplications [multiplex ligation-dependent probe amplification (MLPA), polymerase chain reaction (PCR)] and cognitive status assessment (Wechsler Intelligence Scale for Children, Brunet-Lezine scale, Vineland-Doll scale). In 37 patients with an estimated full scale intelligence quotient (FSIQ), six (16.22%) had borderline intelligence (70<FSIQ ≤85), while seven (18.92%) were intellectually impaired (FSIQ <70). The FSIQ was not associated with proximal and distal mutations when boundaries were set at exons 30 and 45. However, FSIQ was statistically significantly associated with mutation location when we assumed their functional consequence on dystrophin isoforms and when mutations in the 5′-untranslated region (5′UTR) of Dp140 (exons 45–50) were assigned to affect only Dp427 and Dp260. Mutations affecting Dp140 and Dp71/Dp40 have been associated with more frequent and more severe cognitive impairment. Finally, the same classification of mutations explained the greater proportion of FSIQ variability associated with cumulative loss of dystrophin isoforms. In conclusion, cumulative loss of dystrophin isoforms increases the risk of intellectual impairment in DMD and characterizing the genotype can define necessity of early cognitive interventions in DMD patients.

Detection of dystrophin gene mutation in a Chinese pedigree affected with Duchenne muscular dystrophy

To identify mutations of dystrophin gene in a Chinese pedigree affected with Duchenne muscular dystrophy (DMD). Clinical data from the pedigree was collected. Subsequently, polymerase chain reaction and DNA sequencing analysis were applied to detect the potential mutations. Restriction enzyme digestion was carried out to determine whether the mutation was present in 118 healthy controls. Clustal software was applied for analyzing the conservation of altered amino acids. DNA sequencing analysis has identified a heterozygous missense mutation c.7578G>C (p.Gln2526His) mutation in exon 52 of the dystrophin gene in the proband and his mother. The same mutation was absent in the 118 healthy controls. Restriction enzyme digestion has confirmed above result. Clustal analysis indicated that the altered amino acid is highly conserved in mammals. The results revealed a novel missense mutation (c.7578G>C) of the dystrophin gene in DMD patients.

204th ENMC International Workshop on Biomarkers in Duchenne Muscular Dystrophy 24–26 January 2014, Naarden, The Netherlands

This report reflects the state-of-the-art and future directions of basic and clinical research into biomarkers for Duchenne Muscular Dystrophy (DMD), as discussed during the 204th ENMC workshop in Naarden, from January 24–26, 2014. Biomarkers have been defined as cellular, biochemical, molecular alterations or biological characteristics that are measurable in biological material as indicator of normal biological or pathogenic processes [1]. Biomarkers may be used in differential and early diagnosis, and in monitoring of disease progression, regression, or therapeutic responses. Duchenne Muscular Dystrophy (DMD) is a severe hereditary muscle disorder due dystrophin gene mutations and presenting with variable clinical severity [2]. Recently novel experimental drugs have been developed for DMD and several trials are ongoing, raising the urgent need of having fine tools for measuring trial outcomes as well as for optimizing the selection of eligible patients [3, 4]. The use of clinical parameters measuring muscle strength and function is limited due to their dependence on motivation, large intra-individual variability, lack of linear relationship between the 2 and slow response time [5]. Conversely, molecular biomarkers may show earlier response to treatment and reflect the different pathophysiological aspects of the disease [6]. The use of biomarker panels for the diagnosis, prognosis, and monitoring of DMD (and more in general of rare chronic neuromuscular disorders or NMDs) as well as to guide the choice of therapeutic regimens may significantly improve the current clinical practice, by facilitating the evaluation of emerging therapies in drug trials and their regulatory approval. On the other hand, during the drug development process, the availability of a biomarker able to predict drug response and/or occurrence of adverse events could be of utmost importance and could also reduce the costs of the drug development [7]. Biomarkers can also serve for patient stratification and selection of appropriate subjects for clinical trials. Biomarkers can therefore have a positive impact on the economical load, patients’ care and novel therapies. Since the field of biomarker development is in a state of flux and given the need for biomarkers for DMD, an ENMC workshop took place in Naarden on the 23–25th of January 2014. It aimed at sharing data and results on biomarker discovery and validation for DMD, as well as to define strategies to implement biomarker discovery and use in DMD clinical trials. 27 people attended, including clinicians, researchers, drug companies, parents association, and an expert associated to EMA.

G.P.82: Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle

Duchenne muscular dystrophy (DMD) is caused by mutations in the X-linked dystrophin (DMD) gene. The absence of dystrophin protein leads to progressive muscle weakness and wasting, disability and death. Existing animal models have been instrumental to understand the pathophysiology of DMD, but have limitations related to the type of mutation, the clinical phenotype, and the predictive value for molecular therapies. To establish a tailored large animal model of DMD, we deleted DMD exon 52 in male pig cells by gene targeting and generated offspring by nuclear transfer. DMD pigs exhibit absence of dystrophin in skeletal muscles, increased serum creatine kinase levels, progressive dystrophic changes of skeletal muscles, impaired mobility, muscle weakness, and a maximum life span of 3 months due to respiratory impairment. These findings render the DMD mutant pig a promising model for testing targeted genetic treatment approaches, such as exon skipping which partially restored in-frame dystrophin gene expression in our model. Regarding recent disappointing results of exon skipping in human DMD clinical trials, a valid animal model will be warranted.

G.P.2: Clinical profile of pediatric patients with Becker Muscular Dystrophy (BMD)

Antisense oligonucleotide (AON)-induced exon skipping in Duchenne muscular dystrophy (DMD) patients aim to restore out of frame dystrophin gene mutations to in-frame mutations with resultant dystrophin production and a BMD phenotype. BMD occurs in 1 in 20000 males and is associated with partially functional truncated dystrophin protein. Pending on the amount of truncated dystrophin, the clinical phenotype of BMD patients vary from those with loss of ambulation in the mid-teens to patients with normal skeletal motor function in late adulthood. Natural history data of pediatric BMD patients would provide useful clinical information regarding the potential endpoints of therapeutic efficacy of exon skipping treatment trials. To characterize the clinical profile of pediatric patients with Becker Muscular Dystrophy (BMD) IRB approved retrospective case series' review of BMD patients Clinical profiles of BMD patients: 48 males with clinical and genetic/or histologic diagnosis of Becker's Muscular Dystrophy. Patient characteristics: Mean age at diagnosis – 5.4years (0.75–12years); mean CK at time of diagnosis – 8036 (161–40,000); Presenting symptom(s) – elevated LFTs (10/48), gross motor delay (9/48), hypotonia (6/48), exercise intolerance/myalgias (15/48), abnormal walking/running gait (17/48), family history (6/48), incidental lab finding (9/48), fatigue (1/48); Indices of muscle mass: Pelvic/thigh Muscle MRI – (<i>n</i>=33) normal study to trace fatty infiltration – 16, mild-to-moderate fatty infiltration – 11, diffuse/significant – 6; mean serum creatinine – 0.41 (0.1–0.8); DEXA mean % lean muscle mass – 70 (51–82); Bone health: mean DEXA distal femur R1 <i>z</i>-score – 0.75 (2.7–3.8); Cardiac function: mean left ventricular ejection fraction (%) – 63 (50–77); Motor status at last follow-up – normal function – 24, stable, near-normal function – 19, stable, abnormal function – 2, decline in motor function – 1, lost to follow-up – 2.

MLPA Application in Clinical Diagnosis of DMD/BMD in Shanghai.

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked recessive disorders caused by mutation in dystrophin gene. We reported 3-year clinic experience from a single hospital in Shanghai using multiplex ligation dependent probe amplification (MLPA) assay to detect DMD mutations. Four hundred and fifty-one males and 184 females, who were clinically diagnosed as DMD/BMD patients or carriers at our hospital's outpatient clinic, were collected and performed with MLPA to detect DMD gene mutations. Seventeen novel mutation points not reported in the Leiden Muscular Dystrophy pages were identified in this study. We found that the most frequent deletion spots ranged from exon45 to exon52, and exon2, exon19 were the two most frequently detected duplication spots. The results of our study confirmed MLPA as an efficient clinical method for detecting DMD gene mutations in DMD/BMD patients. Single exon mutation detected by MLPA should be verified by other methods, and we should emphasize that only precise clinical molecular diagnosis can lead to the feasibility of prenatal diagnosis.

Characterization of muscular involvement in patients with Duchenne muscular dystrophy by magnetic resonance imaging

To study the order and degree of muscular affection in patients with Duchenne muscular dystrophy (DMD) during the course of disease. Multiplex ligation dependent probe amplification (MLPA) was used to detect potential mutation of dystrophin gene. Magnetic resonance imaging (MRI) was used to scan the anteromedial aspect of thigh muscles. All of the 6 patients were found to have deletion or duplication mutations. The order of affection has been gluteus maximus, adductor magnus, quadriceps femoris, rectus femoris and biceps muscle of the thigh, while semimembranous muscle, semitendinosus, sartorius muscle and musculus gracilis are selectively affected and in a decreasing order. MRI can reflect the order, extent and degree of skeletal muscle involvement in patients with DMD, and can reflect pathological changes of damaged skeletal muscle at each stage, which may provide an important means for patient examination and diagnosis. No apparent correlation between the severity of disease and the nature of mutations was noted.

Muscle Disease

On the basis of strong research evidence, Duchenne muscular dystrophy (DMD), the most common severe childhood form of muscular dystrophy, is an X-linked recessive disorder caused by out-of-frame mutations of the dystrophin gene. Thus, it is classified asa dystrophinopathy. The disease onset is before age 5 years. Patients with DMD present with progressive symmetrical limb-girdle muscle weakness and become wheelchair dependent after age 12 years. (2)(3). On the basis of some research evidence,cardiomyopathy and congestive heart failure are usually seen in the late teens in patients with DMD. Progressive scoliosis and respiratory in sufficiency often develop once wheelchair dependency occurs. Respiratory failure and cardiomyopathy are common causes of death, and few survive beyond the third decade of life. (2)(3)(4)(5)(6)(7). On the basis of some research evidence, prednisone at 0.75 mg/kg daily (maximum dose, 40 mg/d) or deflazacort at 0.9 mg/kg daily (maximum dose, 39 mg/d), a derivative of prednisolone (not available in the United States), as a single morning dose is recommended for DMD patients older than 5 years, which may prolong independent walking from a few months to 2 years. (2)(3)(16)(17). Based on some research evidence, treatment with angiotensin-converting enzyme inhibitors, b-blockers, and diuretics has been reported to be beneficial in DMD patients with cardiac abnormalities. (2)(3)(5)(18). Based on expert opinion, children with muscle weakness and increased serum creatine kinase levels may be associated with either genetic or acquired muscle disorders (Tables 1 and 3). (14)(15)

Association between specific dystrophin gene mutations and myocardial fibrosis by cardiovascular magnetic resonance imaging in patients with Duchenne and Becker muscular dystrophy

Background Duchenne (DMD) and Becker (BMD) muscular dystrophies (MD) are allelic X-linked recessive disorders, caused by mutation of the dystrophin gene located at locus Xp21 that consists of 79 exons, characterized by progressive skeletal muscle degeneration and replacement by fibro fatty tissue. Dystrophin is a sarcolemal protein that links the cytoskeleton to the basal lamina and is essential for maintenance of the muscular membrane integrity during muscular contraction. Cardiac involvement is frequent, 70 - 80% of patients, and often develops clinically silent, without any evident early clinical signs. CMR can identify myocardial fibrosis (MF) and may be useful for detecting the early stages of cardiomyopathy in MD. In a previous study, DNA analyses in 47 pts with DMD revealed significant association between dilated cardiomyopathy (DCM) and specific exons and possible protection against DCM by other exons. The association between specific exons mutation of the dystrophin gene and myocardial fibrosis is until unknown.

What got us here will not get us there

This is an exciting yet trying time for people living with neuromuscular diseases and those who care for them. The exciting aspect is that at last the advances in genetics and translational research have resulted in clinical trials testing novel compounds. Examples include the exon-skipping approaches in Duchenne Muscular Dystrophy (DMD). Resulting from a sophisticated understanding of the consequences of dystrophin gene mutations, ASOs were developed to help transform the “garbled” message resulting from a disrupted reading frame into a “translatable”, in-frame, message. This rational and targeted approach worked in pre-clinical studies, but it yet has to confirm its efficacy in patients. The trying aspect illustrated by this example is that it has taken over 10 years to get from pre-clinical proofof principle studies to clinical trials. Some of this is the inevitable development time needed for candidate selection, lead optimization, pre-clinical toxicology, and manufacturing issues. However, an additional source of delay may be the limited tools and data available for trial planning: The available methodology to measure dystrophin in muscle biopsies may not be sensitive enough, and may show too much variation due to sampling or differences in laboratory measurements. Similarly, existing clinical outcome measures have limitations in terms of floor and ceiling effects, in terms of their applicability to

What Can We Learn From Clinical Trials of Exon Skipping for DMD?

On 20 September 2013, GlaxoSmithKline (GSK) and Prosensa announced that GSK's Phase III clinical trial ({"type":"clinical-trial","attrs":{"text":"NCT01254019","term_id":"NCT01254019"}}NCT01254019) of Drisapersen, an exon skipping drug for Duchenne muscular dystrophy (DMD), failed to meet the primary endpoint of a statistically significant improvement in the 6 Minute Walking Distance Test (6MWT) compared to placebo.1 On 12 November 2013, Sarepta Therapeutics announced that the US Food and Drug Administration (FDA) has considered its application for accelerated approval of Eteplirsen as a DMD drug to be premature.2 The news came as a great disappointment to the scientific community and more specifically to the families and foundations that had followed the trail of research articles and press announcements. Moreover, the clinical trial results and the FDA's view of the relationship between the dystrophin biomarker and functional endpoint of 6MWT in clinic will have a far reaching impact beyond exon skipping therapy in DMD.

Exome sequencing discovered a novel splice donor mutation in dystrophin gene

Objective To detect subtle mutations in the dystrophin gene by exome capturing using second-generation sequencing technique.Methods Exome capturing using second-generation sequencing technique was used to detect mutations of dystrophin gene in a patient with typical clinical manifestations of Duchenne muscular dystrophy(DMD),but without deletions or duplications in the dystrophin gene.The mutations were verified by Sanger sequencing,and bioinformatics was employed to predict its influence on the coding.The patient's mother and 100 healthy volunteers were taken as controls.Results A base change in the first base of intron 50(GC) was found in dystrophin gene of the patient,and his mother was heterozygosis at the same site.Bioinformatics predicted that the 5 donor splicing site of intron 50 would disappear due to this base change, which would alter the amino acid sequence at the C terminal of corresponding peptide and result in the appearance of premature termination codon.Sanger sequencing confirmed that the base change was a novel pathogenic mutation in the dystrophin gene, and it was absent in normal controls.Conclusion It is demonstrated that exome sequencing technique can effectively detect the subtle mutations in the dystrophin gene,which may contribute to better molecular diagnosis of DMD.

Decrease in prosaposin in the Dystrophic mdx mouse brain.

BackgroundDuchenne muscular dystrophy caused by a mutation in the X-linked dystrophin gene induces metabolic and structural disorders in the brain. A lack of dystrophin in brain structures is involved in impaired cognitive function. Prosaposin (PS), a neurotrophic factor, is abundant in the choroid plexus and various brain regions. We investigated whether PS serves as a link between dystrophin loss and gross and/or ultrastructural brain abnormalities.Methodology/Principal FindingsThe distribution of PS in the brains of juvenile and adult mdx mice was investigated by immunochemistry, Western blotting, and in situ hybridization. Immunochemistry revealed lower levels of PS in the cytoplasm of neurons of the cerebral cortex, hippocampus, cerebellum, and choroid plexus in mdx mice. Western blotting confirmed that PS levels were lower in these brain regions in both juveniles and adults. Even with low PS production in the choroids plexus, there was no significant PS decrease in cerebrospinal fluid (CSF). In situ hybridization revealed that the primary form of PS mRNA in both normal and mdx mice was Pro+9, a secretory-type PS, and the hybridization signals for Pro+9 in the above-mentioned brain regions were weaker in mdx mice than in normal mice. We also investigated mitogen-activated protein kinase signalling. Stronger activation of ERK1/2 was observed in mdx mice, ERK1/2 activity was positively correlated with PS activity, and exogenous PS18 stimulated both p-ERK1/2 and PS in SH-SY5Y cells.Conclusions/SignificanceLow levels of PS and its receptors suggest the participation of PS in some pathological changes in the brains of mdx mice.

Removing the Immune Response From Muscular Dystrophy Research

Immune responses against exogenous cells, vectors, and proteins have emerged as a significant barrier limiting development of therapies for inherited disorders. This is especially true when testing human cells and genes in animal models of disease. In this issue of Molecular Therapy, Vallese et al.1 describe an improved immunodeficient mouse model for Duchenne muscular dystrophy (DMD) that should facilitate the development of therapies. The authors show that these mice enable extensive testing of human stem cells and methods of delivery without immune-mediated rejection of donor cells. The utility of this model is further enhanced by use of a specific mutant allele that eliminates background dystrophin expression in muscle tissues, providing a sensitive platform for monitoring cell engraftment and exogenous gene expression. As such, the model could accelerate development of interventions based on both stem cell transplantation and genetic manipulation.

Anti-dystrophin T cell responses in Duchenne muscular dystrophy: prevalence and a glucocorticoid treatment effect.

Duchenne muscular dystrophy (DMD) typically occurs as a result of truncating mutations in the DMD gene that result in a lack of expression of the dystrophin protein in muscle fibers. Various therapies under development are directed toward restoring dystrophin expression at the subsarcolemmal membrane, including gene transfer. In a trial of intramuscular adeno-associated virus (AAV)-mediated delivery of a therapeutic minidystrophin construct, we identified in two of six subjects the presence of a population of T cells that had been primed to recognize dystrophin epitopes before transgene delivery. As the presence of preexisting T cell immunity may have a significant effect on the success of therapeutic approaches for restoring dystrophin, we sought to determine the prevalence of such immunity within a DMD cohort from our Muscular Dystrophy Association clinic. Dystrophin-specific T cell immunity was evaluated in subjects with DMD who were either receiving the glucocorticoid steroid prednisone (n=24) or deflazacort (n=29), or who were not receiving steroids (n=17), as well as from normal age-matched control subjects (n=21). We demonstrate that increasing age correlates with an increased risk for the presence of anti-dystrophin T cell immunity, and that treatment with either corticosteroid decreases risk compared with no treatment, suggesting that steroid therapy in part may derive some of its benefit through modulation of T cell responses. The frequency of dystrophin-specific T cells detected by enzyme-linked immunospot assay was lower in subjects treated with deflazacort versus prednisone, despite similar overall corticosteroid exposure, suggesting that the effects of the two corticosteroids may not be identical in patients with DMD. T cells targeted epitopes upstream and downstream of the dystrophin gene mutation and involved the CD4⁺ helper and/or CD8⁺ cytotoxic subsets. Our data confirm the presence of preexisting circulating T cell immunity to dystrophin in a sizable proportion of patients with DMD, and emphasize the need to consider this in the design and interpretation of clinical gene therapy trials.