Quantitative NMRI and NMRS identify augmented disease progression after loss of ambulation in forearms of boys with Duchenne muscular dystrophy.

Quantitative NMRI and (31)P NMRS indices are reported in the forearms of 24 patients with Duchenne muscular dystrophy (DMD) (6-18 years, 14 non-ambulant) amenable to exon 53 skipping therapy and in 12 age-matched male controls (CONT). Examinations carried out at 3 T comprised multi-slice 17-echo measurements of muscle water T2 and heterogeneity, three-point Dixon imaging of fat fraction in flexor and extensor muscles (FLEX, EXT), and non-localised spectroscopy of phosphate metabolites. We studied four imaging indices, eight metabolic ratios combining ATP, phosphocreatine, phosphomonoesters and phosphodiesters, the cytosolic inorganic phosphate (Pia ) and an alkaline (Pib) pool present in dystrophic muscle, and average pH. All indices differed between DMD and CONT, except for muscle water T2 . Measurements were outside the 95th percentile of age-matched CONT values in over 65% of cases for percentage fat signal (%F), and in 78-100% of cases for all spectroscopic indices. T2 was elevated in one-third of FLEX measurements, whereas %pixels > 39 ms and T2 heterogeneity were abnormal in one-half of the examinations. The FLEX muscles had higher fat infiltration and T2 than EXT muscle groups. All indices, except pH, correlated with patient age, although the correlation was negative for T2 . However, in non-ambulant patients, the correlation with years since loss of ambulation was stronger than the correlation with age, and the slope of evolution per year was steeper after loss of ambulation. All indices except Pi/gATP differed between ambulant and non-ambulant patients; however, T2 and %pixels > 39 ms were highest in ambulant patients, possibly owing to the greater extent of inflammatory processes earlier in the disease. All other indices were worse in non-ambulant subjects. Quantitative measurements obtained from patients at different disease stages covered a broad range of abnormalities that evolved with the disease, and metabolic indices were up to 10-fold above normal from the onset, thus establishing a variety of potential markers for future therapy.

Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.

A phase I trial of adeno-associated virus serotype 1-γ-sarcoglycan gene therapy for limb girdle muscular dystrophy type 2C.

γ-Sarcoglycanopathy or limb girdle muscular dystrophy type 2C is an untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene. Nine non-ambulatory patients (two males, seven females, mean age 27 years; range 16-38 years) with del525T homozygous mutation of the γ-sarcoglycan gene and no γ-sarcoglycan immunostaining on muscle biopsy were divided into three equal groups to receive three escalating doses of an adeno-associated virus serotype 1 vector expressing the human γ-sarcoglycan gene under the control of the desmin promoter, by local injection into the extensor carpi radialis muscle. The first group received a single injection of 3 × 10(9) viral genomes in 100 µl, the second group received a single injection of 1.5 × 10(10) viral genomes in 100 µl, and the third group received three simultaneous 100-µl injections at the same site, delivering a total dose of 4.5 × 10(10) viral genomes. No serious adverse effects occurred during 6 months of follow-up. All nine patients became adeno-associated virus serotype 1 seropositive and one developed a cytotoxic response to the adeno-associated virus serotype 1 capsid. Thirty days later, immunohistochemical analysis of injected-muscle biopsy specimens showed γ-sarcoglycan expression in all three patients who received the highest dose (4.7-10.5% positively stained fibres), while real-time polymerase chain reaction detected γ-sarcoglycan messenger RNA. In one patient, γ-sarcoglycan protein was detected by western blot. For two other patients who received the low and intermediate doses, discrete levels of γ-sarcoglycan expression (<1% positively stained fibres) were also detectable. Expression of γ-sarcoglycan protein can be induced in patients with limb girdle muscular dystrophy type 2C by adeno-associated virus serotype 1 gene transfer, with no serious adverse effects.

A 10 patient case report on the impact of plasmapheresis upon neutralizing factors against adeno-associated virus (AAV) types 1, 2, 6, and 8.

Adeno-associated viruses (AAV) are small, nonenveloped single-stranded DNA viruses which require helper viruses to facilitate efficient replication. These recombinant viruses are some of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Nevertheless, the presence of humoral responses to the wild-type AAV common among humans is one of the limitations of in vivo transduction efficacy in humans using cognate recombinant vector. In this study, based on the serum samples that we were able to collect from various clinical situations, we studied the impact of one to five plasmapheresis (PP), at 1-5 day intervals on neutralizing factor (NAF) titers specific for AAV types 1, 2, 6, and 8 in seropositive patients with diverse pathologies and immunosuppressor treatments. We show that frequent sessions of PP result in drastic reduction of NAF specific for AAV1, 2, 6, and 8 to undetectable levels or titers <1:5, mainly when initial titers, i.e., before the first PP were ≤1:20. Altogether, these results show that the use of PP and its possible association with pharmacological immunosuppressive treatments may help to design optimal management of seropositive patients for AAV gene therapy treatments.

AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis.

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.

Evidence for long-term efficacy and safety of gene therapy for Wiskott-Aldrich syndrome in preclinical models.

Wiskott-Aldrich Syndrome (WAS) is a life-threatening X-linked disease characterized by immunodeficiency, thrombocytopenia, autoimmunity, and malignancies. Gene therapy could represent a therapeutic option for patients lacking a suitable bone marrow (BM) donor. In this study, we analyzed the long-term outcome of WAS gene therapy mediated by a clinically compatible lentiviral vector (LV) in a large cohort of was(null) mice. We demonstrated stable and full donor engraftment and Wiskott-Aldrich Syndrome protein (WASP) expression in various hematopoietic lineages, up to 12 months after gene therapy. Importantly, we observed a selective advantage for T and B lymphocytes expressing transgenic WASP. T-cell receptor (TCR)-driven T-cell activation, as well as B-cell's ability to migrate in response to CXCL13, was fully restored. Safety was evaluated throughout the long-term follow-up of primary and secondary recipients of WAS gene therapy. WAS gene therapy did not affect the lifespan of treated animals. Both hematopoietic and nonhematopoietic tumors arose, but we excluded the association with gene therapy in all cases. Demonstration of long-term efficacy and safety of WAS gene therapy mediated by a clinically applicable LV is a key step toward the implementation of a gene therapy clinical trial for WAS.

Myostatin Is a Quantifiable Biomarker for Monitoring Pharmaco-gene Therapy in Duchenne Muscular Dystrophy.

Recently, several promising treatments have emerged for neuromuscular disorders, highlighting the need for robust biomarkers for monitoring therapeutic efficacy and maintenance of the therapeutic effect. Several studies have proposed circulating and tissue biomarkers, but none of them has been validated to monitor acute and long-term drug response. We previously described how the myostatin (MSTN) level is naturally downregulated in several neuromuscular diseases, including Duchenne muscular dystrophy (DMD). Here, we show that the dystrophin-deficient Golden Retriever muscular dystrophy (GRMD) dog model also presents an intrinsic loss of Mstn production in muscle. The abnormally low levels of Mstn observed in the GRMD dog puppies at 2 months were partially rescued at both mRNA and protein level after adeno-associated virus (AAV)-microdystrophin treatment in a dose-dependent manner. These results show that circulating Mstn is a robust and reliable quantitative biomarker, capable of measuring a therapeutic response to pharmaco-gene therapy in real time in the neuromuscular system, as well as a quantitative means for non-invasive follow-up of a therapeutic effect. Moreover, a 2-year follow-up also suggests that Mstn could be a longitudinal monitoring tool to follow maintenance or decrease of the therapeutic effect.

An AAV-SGCG Dose-Response Study in a γ-Sarcoglycanopathy Mouse Model in the Context of Mechanical Stress.

Sarcoglycanopathies are rare autosomic limb girdle muscular dystrophies caused by mutations in one of the genes coding for sarcoglycans. Sarcoglycans form a complex, which is an important part of the dystrophin-associated glycoprotein complex and which protects the sarcolemma against muscle contraction-induced damage. Absence of one of the sarcoglycans on the plasma membrane reduces the stability of the whole complex and perturbs muscle fiber membrane integrity. There is currently no curative treatment for any of the sarcoglycanopathies. A first clinical trial to evaluate the safety of a recombinant AAV2/1 vector expressing γ-sarcoglycan using an intramuscular route of administration showed limited expression of the transgene and good tolerance of the approach. In this report, we undertook a dose-effect study in mice to evaluate the efficiency of an AAV2/8-expressing γ-sarcoglycan controlled by a muscle-specific promoter with a systemic mode of administration. We observed a dose-related efficiency with a nearly complete restoration of gamma sarcoglycan (SGCG) expression, histological appearance, biomarker level, and whole-body strength at the highest dose tested. In addition, our data suggest that a high expression threshold level must be achieved for effective protection of the transduced muscle, while a suboptimal transgene expression level might be less protective in the context of mechanical stress.

RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect.

Non-coding uridine-rich small nuclear RNAs (UsnRNAs) have emerged in recent years as effective tools for exon skipping for the treatment of Duchenne muscular dystrophy (DMD), a degenerative muscular genetic disorder. We recently showed the high capacity of a recombinant adeno-associated virus (rAAV)-U7snRNA vector to restore the reading frame of the DMD mRNA in the muscles of DMD dogs. We are now moving toward a phase I/II clinical trial with an rAAV-U7snRNA-E53, carrying an antisense sequence designed to hybridize exon 53 of the human DMD messenger. As observed for genome-editing tools, antisense sequences present a risk of off-target effects, reflecting partial hybridization onto unintended transcripts. To characterize the clinical antisense sequence, we studied its expression and explored the occurrence of its off-target effects in human in vitro models of skeletal muscle and liver. We presented a comprehensive methodology combining RNA sequencing and in silico filtering to analyze off-targets. We showed that U7snRNA-E53 induced the effective exon skipping of the DMD transcript without inducing the notable deregulation of transcripts in human cells, neither at gene expression nor at the mRNA splicing level. Altogether, these results suggest that the use of the rAAV-U7snRNA-E53 vector for exon skipping could be safe in eligible DMD patients.

Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV-microdystrophin gene therapy in DMD patients.

Dystrophin Threshold Level Necessary for Normalization of Neuronal Nitric Oxide Synthase, Inducible Nitric Oxide Synthase, and Ryanodine Receptor-Calcium Release Channel Type 1 Nitrosylation in Golden Retriever Muscular Dystrophy Dystrophinopathy.

At present, the clinically most advanced strategy to treat Duchenne muscular dystrophy (DMD) is the exon-skipping strategy. Whereas antisense oligonucleotide-based clinical trials are underway for DMD, it is essential to determine the dystrophin restoration threshold needed to ensure improvement of muscle physiology at the molecular level. A preclinical trial has been conducted in golden retriever muscular dystrophy (GRMD) dogs treated in a forelimb by locoregional delivery of rAAV8-U7snRNA to promote exon skipping on the canine dystrophin messenger. Here, we exploited rAAV8-U7snRNA-transduced GRMD muscle samples, well characterized for their percentage of dystrophin-positive fibers, with the aim of defining the threshold of dystrophin rescue necessary for normalization of the status of neuronal nitric oxide synthase mu (nNOSµ), inducible nitric oxide synthase (iNOS), and ryanodine receptor-calcium release channel type 1 (RyR1), crucial actors for efficient contractile function. Results showed that restoration of dystrophin in 40% of muscle fibers is needed to decrease abnormal cytosolic nNOSµ expression and to reduce overexpression of iNOS, these two parameters leading to a reduction in the NO level in the muscle fibers. Furthermore, the same percentage of dystrophin-positive fibers of 40% was associated with the normalization of RyR1 nitrosylation status and with stabilization of the RyR1-calstabin1 complex that is required to facilitate coupled gating. We concluded that a minimal threshold of 40% of dystrophin-positive fibers is necessary for the reinstatement of central proteins needed for proper muscle contractile function, and thus identified a rate of dystrophin expression significantly improving, at the molecular level, the dystrophic muscle physiology.

Non-Ambulant Duchenne Patients Theoretically Treatable by Exon 53 Skipping have Severe Phenotype.

BACKGROUND: Exon skipping therapy is an emerging approach in Duchenne Muscular Dystrophy (DMD). Antisense oligonucleotides that induce skipping of exon 51, 44, 45, or 53 are currently being evaluated in clinical trials. These trials were designed on the basis of data available in general DMD population. OBJECTIVES: Our objective was to compare the clinical and functional statuses of non-ambulant DMD patients theoretically treatable by exon 53 skipping and of DMD patients with other mutations. METHODS: We first compared fifteen non-ambulant DMD patients carrying deletions theoretically treatable by exon 53 skipping (DMD-53) with fifteen closely age-matched DMD patients with mutations not treatable by exon 53 skipping (DMD-all-non-53) then with fifteen DMD patients carrying deletions not treatable by exon 53 skipping (DMD-del-non-53). RESULTS: We found that DMD-53 patients had a lower left ventricular ejection fraction, more contractures and they tend to have weaker grips and pinch strengths than other DMD patients. DMD-53 patients lost ambulation significantly younger than other DMD patients. This result was confirmed by comparing ages at loss of ambulation in all non-ambulant DMD patients of the DMD cohort identified in a molecular diagnostic lab. CONCLUSIONS: These prospective and retrospective data demonstrate that DMD-53 patients have clinically more severe phenotypes than other DMD patients.

Distinctive serum miRNA profile in mouse models of striated muscular pathologies.

Biomarkers are critically important for disease diagnosis and monitoring. In particular, close monitoring of disease evolution is eminently required for the evaluation of therapeutic treatments. Classical monitoring methods in muscular dystrophies are largely based on histological and molecular analyses of muscle biopsies. Such biopsies are invasive and therefore difficult to obtain. The serum protein creatine kinase is a useful biomarker, which is however not specific for a given pathology and correlates poorly with the severity or course of the muscular pathology. The aim of the present study was the systematic evaluation of serum microRNAs (miRNAs) as biomarkers in striated muscle pathologies. Mouse models for five striated muscle pathologies were investigated: Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy type 2D (LGMD2D), limb-girdle muscular dystrophy type 2C (LGMD2C), Emery-Dreifuss muscular dystrophy (EDMD) and hypertrophic cardiomyopathy (HCM). Two-step RT-qPCR methodology was elaborated, using two different RT-qPCR miRNA quantification technologies. We identified miRNA modulation in the serum of all the five mouse models. The most highly dysregulated serum miRNAs were found to be commonly upregulated in DMD, LGMD2D and LGMD2C mouse models, which all exhibit massive destruction of striated muscle tissues. Some of these miRNAs were down rather than upregulated in the EDMD mice, a model without massive myofiber destruction. The dysregulated miRNAs identified in the HCM model were different, with the exception of one dysregulated miRNA common to all pathologies. Importantly, a specific and distinctive circulating miRNA profile was identified for each studied pathological mouse model. The differential expression of a few dysregulated miRNAs in the DMD mice was further evaluated in DMD patients, providing new candidates of circulating miRNA biomarkers for DMD.

Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors.

Adeno-associated viruses (AAVs) are small, nonenveloped single-stranded DNA viruses that require helper viruses to facilitate efficient replication. Despite the presence of humoral responses to the wild-type AAV in humans, AAV remains one of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Characterization of the IgG subclass responses to AAV and study of the prevalence of both IgG and neutralizing factors to AAV types 1, 2, 5, 6, 8, and 9 in the human population are of importance for the development of new strategies to overcome these immune responses. Natural exposure to AAV types 1, 2, 5, 6, 8, and 9 can result in the production of antibodies from all four IgG subclasses, with a predominant IgG1 response and low IgG2, IgG3, and IgG4 responses. Prevalences of anti-AAV1 and -AAV2 total IgG determined by enzyme-linked immunosorbent assay were higher (67 and 72%) than those of anti-AAV5 (40%), anti-AAV6 (46%), anti-AAV8 (38%), and anti-AAV9 (47%). Furthermore, data showed that cross-reactions are important. The two highest neutralizing factor seroprevalences were observed for AAV2 (59%) and AAV1 (50.5%) and the lowest were observed for AAV8 (19%) and AAV5 (3.2%). Vectors based on AAV5, AAV8, and AAV9 may have an advantage for gene therapy in humans. Furthermore, among individuals seropositive for AAV5, AAV8, and AAV9, about 70-100% present low titers. Better characterization of the preexisting humoral responses to the AAV capsid and cross-reactivity will allow development of new strategies to circumvent AAV acquired immune responses.