Adeline Vulin

Muscle hypertrophy driven by myostatin blockade does not require stem/precursor-cell activity

Myostatin, a member of the TGF-β family, has been identified as a powerful inhibitor of muscle growth. Absence or blockade of myostatin induces massive skeletal muscle hypertrophy that is widely attributed to proliferation of the population of muscle fiber-associated satellite cells that have been identified as the principle source of new muscle tissue during growth and regeneration. Postnatal blockade of myostatin has been proposed as a basis for therapeutic strategies to combat muscle loss in genetic and acquired myopathies. But this approach, according to the accepted mechanism, would raise the threat of premature exhaustion of the pool of satellite cells and eventual failure of muscle regeneration. Here, we show that hypertrophy in the absence of myostatin involves little or no input from satellite cells. Hypertrophic fibers contain no more myonuclei or satellite cells and myostatin had no significant effect on satellite cell proliferation in vitro, while expression of myostatin receptors dropped to the limits of detectability in postnatal satellite cells. Moreover, hypertrophy of dystrophic muscle arising from myostatin blockade was achieved without any apparent enhancement of contribution of myonuclei from satellite cells. These findings contradict the accepted model of myostatin-based control of size of postnatal muscle and reorient fundamental investigations away from the mechanisms that control satellite cell proliferation and toward those that increase myonuclear domain, by modulating synthesis and turnover of structural muscle fiber proteins. It predicts too that any benefits of myostatin blockade in chronic myopathies are unlikely to impose any extra stress on the satellite cells.

Muscle Function Recovery in Golden Retriever Muscular Dystrophy After AAV1-U7 Exon Skipping

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.

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.

Long-term Restoration of Cardiac Dystrophin Expression in Golden Retriever Muscular Dystrophy Following rAAV6-mediated Exon Skipping

Although restoration of dystrophin expression via exon skipping in both cardiac and skeletal muscle has been successfully demonstrated in the mdx mouse, restoration of cardiac dystrophin expression in large animal models of Duchenne muscular dystrophy (DMD) has proven to be a challenge. In large animals, investigators have focused on using intravenous injection of antisense oligonucleotides (AO) to mediate exon skipping. In this study, we sought to optimize restoration of cardiac dystrophin expression in the golden retriever muscular dystrophy (GRMD) model using percutaneous transendocardial delivery of recombinant AAV6 (rAAV6) to deliver a modified U7 small nuclear RNA (snRNA) carrying antisense sequence to target the exon splicing enhancers of exons 6 and 8 and correct the disrupted reading frame. We demonstrate restoration of cardiac dystrophin expression at 13 months confirmed by reverse transcription-PCR (RT-PCR) and immunoblot as well as membrane localization by immunohistochemistry. This was accompanied by improved cardiac function as assessed by cardiac magnetic resonance imaging (MRI). Percutaneous transendocardial delivery of rAAV6 expressing a modified U7 exon skipping construct is a safe, effective method for restoration of dystrophin expression and improvement of cardiac function in the GRMD canine and may be easily translatable to human DMD patients.

Immortalized Skin Fibroblasts Expressing Conditional MyoD as a Renewable and Reliable Source of Converted Human Muscle Cells to Assess Therapeutic Strategies for Muscular Dystrophies: Validation of an Exon-Skipping Approach to Restore Dystrophin in Duchenne Muscular Dystrophy Cells

Abstract Numerous strategies are under development for the correction of deleterious effects of mutations in muscular dystrophies, and these strategies must be validated in compelling models. Cellular models seem straightforward to set up; however, the proliferative capacity of muscle cells isolated from dystrophic patients is limited, and in addition it is difficult to envisage the use of large muscle biopsies from patients to obtain enough cells for ex vivo assessments. To overcome these problems, we have devised a strategy to obtain, from a patient with Duchenne muscular dystrophy (DMD), an inexhaustible source of myogenic progenitor cells with a deletion of exons 49 and 50 in the dystrophin gene. Starting material consisted of dermal fibroblasts isolated from a skin biopsy taken in a noninvasive way. These fibroblasts were first immortalized by telomerase gene transfer. Subsequent cell lines were converted into myogenic cells by means of a lentiviral vector encoding an inducible MyoD construct. Before myogenic induction, engineered DMD fibroblasts were able to proliferate infinitely. Under induction conditions, they were converted into myogenic cells, which differentiated into large multinucleated myotubes. We used these DMD fibroblast cell lines to assess dystrophin rescue by using engineered U7 small nuclear RNAs harboring antisense sequences required to restore an in-frame dystrophin mRNA by skipping exon 51. Further molecular analyses showed dystrophin rescue ex vivo as well as in vivo after engrafting of treated cells into regenerating muscles in immunodeficient mice.

Dystrophin quantification: Biological and translational research implications

Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval.

Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject–derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5′ exons of DMD.Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. This novel isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. IRES activity is confirmed in patient muscle by both peptide sequencing and ribosome profiling. Generation of a truncated reading frame upstream of the IRES by exon skipping leads to synthesis of a functional N-truncated isoform in both patient-derived cell lines and in a new DMD mouse model, where expression protects muscle from contraction-induced injury and corrects muscle force to the same level as control mice. These results support a novel therapeutic approach for patients with mutations within the 5’ exons of DMD.

Human epidermal stem cells: Role in adverse skin reactions and carcinogenesis from radiation

In human skin, keratinopoiesis is based on a functional hierarchy among keratinocytes, with rare slow-cycling stem cells responsible for the long-term maintenance of the tissue through their self-renewal potential, and more differentiated daughter progenitor cells actively cycling to permit epidermal renewal and turn-over every month. Skin is a radio-responsive tissue, developing all types of radiation damage and pathologies, including early tissue reactions such as dysplasia and denudation in epidermis, and later fibrosis in the dermis and acanthosis in epidermis, with the TGF-beta 1 pathway as a known master switch. Also there is a risk of basal cell carcinoma, which arises from epidermal keratinocytes, notably after oncogenic events in PTCH1 or TP53 genes. This review will cover the mechanisms of adverse human skin reactions and carcinogenesis after various types of exposures to ionizing radiation, with comparison with animal data when necessary, and will discuss the possible role of stem cells and their progeny in the development of these disorders. The main endpoints presented are basal cell intrinsic radiosensitivity, genomic stability, individual factors of risk, dose specific responses, major molecular pathways involved and the cellular origin of skin reactions and cancer. Although major advances have been obtained in recent years, the precise implications of epidermal stem cells and their progeny in these processes are not yet fully characterized.

Corrigendum: Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice (Nature Medicine (2014))

Nat Med. 20, 992–1000 (2014); doi:10.1038/nm.3628; corrected 25 August 2014; corrected after print 13 March 2015 In the version of this article initially published, three participants of the study were not included as co-authors. Also, one of the individuals mentioned in the Acknowledgments section of the report was incorrectly included and thus has been removed at their request, and the name of another individual mentioned in the Acknowledgments was originally misspelled (“Fabbri” should have been “Fabris”).

Erratum: Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice: (Nature Medicine (2014) 20 (992-1000) DOI:10.1038/nm.3628)

In the version of this article initially published online, the third sentence of the Abstract read “Gene expression analysis identified higher expression of JAK-STAT signaling targets in 3-week-old relative to 18-month-old mice,” when it should have read “Gene expression analysis identified higher expression of JAK-STAT signaling targets in 18-month-old relative to 3-week-old mice.” The error has been corrected for the print, PDF and HTML versions of this article.