In Silico Structural Prediction for the Generation of Novel Performant Midi-Dystrophins Based on Intein-Mediated Dual AAV Approach.

Duchenne Muscular Dystrophy (DMD) is a pediatric disorder characterized by progressive muscle degeneration and premature death, and has no current cure. The current, most promising therapeutic avenue is based on gene replacement mediated by adeno-associated viruses (AAVs) using a shortened, but still functional, version of dystrophin, known as micro-dystrophin (µDys), to fit AAV capacity. The limited improvements observed in clinical trials suggest a sub-optimal performance of µDys in the human context that could be due to the lack of key domains in the protein.

Correction of exon 2, exon 2-9 and exons 8-9 duplications in DMD patient myogenic cells by a single CRISPR/Cas9 system.

Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2-9, and exons 8-9 in the DMD gene.

An engineered AAV targeting integrin alpha V beta 6 presents improved myotropism across species.

Current adeno-associated virus (AAV) gene therapy using nature-derived AAVs is limited by non-optimal tissue targeting. In the treatment of muscular diseases (MD), high doses are often required but can lead to severe adverse effects. Here, we rationally design an AAV capsid that specifically targets skeletal muscle to lower treatment doses.

Late Neolithic collective burial reveals admixture dynamics during the third millennium BCE and the shaping of the European genome.

The third millennium BCE was a pivotal period of profound cultural and genomic transformations in Europe associated with migrations from the Pontic-Caspian steppe, which shaped the ancestry patterns in the present-day European genome. We performed a high-resolution whole-genome analysis including haplotype phasing of seven individuals of a collective burial from ~2500 cal BCE and of a Bell Beaker individual from ~2300 cal BCE in the Paris Basin in France. The collective burial revealed the arrival in real time of steppe ancestry in France.

Basic notions about gene therapy from the nucleic acid perspective and applications in a pediatric disease: Duchenne muscular dystrophy.

Gene therapy involves the introduction of genetic material into cells as a therapeutic molecule to cure a disease. Through the transfer of specific nucleic acid to the target tissue, gene expression can be downregulated, augmented, or corrected thanks to the nucleic acid sequence as a support of gene expression. This is achieved through molecular interactions according to the sequence arrangement or the secondary structure of the molecules or through their catalytic properties.

CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy.

Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects leading to the deficiency of α-DG glycosylation, essential to membrane integrity. Recombinant adeno-associated viral vector (rAAV) gene therapy offers great therapeutic promise for such neuromuscular disorders. Pre-clinical studies have paved the way for a phase 1/2 clinical trial aiming to evaluate the safety and efficacy of FKRP gene therapy in LGMDR9 patients.

Modeling Sarcoglycanopathy in .

Sarcoglycanopathies, also known as limb girdle muscular dystrophy 3-6, are rare muscular dystrophies characterized, although heterogeneous, by high disability, with patients often wheelchair-bound by late adolescence and frequently developing respiratory and cardiac problems. These diseases are currently incurable, emphasizing the importance of effective treatment strategies and the necessity of animal models for drug screening and therapeutic verification. Using the CRISPR/Cas9 genome editing technique, we generated and characterized δ-sarcoglycan and β-sarcoglycan knockout zebrafish lines, which presented a progressive disease phenotype that worsened from a mild larval stage to distinct myopathic features in adulthood.

Assessment of Therapeutic Potential of a Dual AAV Approach for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a yet incurable rare genetic disease that affects the skeletal and cardiac muscles, leading to progressive muscle wasting and premature death. DMD is caused by the lack of dystrophin, a muscle protein essential for the biochemical support and integrity of muscle fibers. Gene replacement strategies for Duchenne muscular dystrophy (DMD) employing the adeno-associated virus (AAV) face the challenge imposed by the limited packaging capacity of AAV, only allowing the accommodation of a short version of dystrophin (µDys) that is still far removed from correcting human disease.

Context-induced renewal of passive but not active coping behaviours in the shock-probe defensive burying task.

Renewal is the return of extinguished responding after removal from the extinction context. Renewal has been extensively studied using classical aversive conditioning procedures that measure a passive freezing response to an aversive conditioned stimulus. However, coping responses to aversive stimuli are complex and can be reflected in passive and active behaviours.

Clinical and functional characterization of a long survivor congenital titinopathy patient with a novel metatranscript-only titin variant.

Congenital titinopathies are an emerging group of a potentially severe form of congenital myopathies caused by biallelic mutations in titin, encoding the largest existing human protein involved in the formation and stability of sarcomeres. In this study we describe a patient with a congenital myopathy characterized by multiple contractures, a rigid spine, non progressive muscular weakness, and a novel homozygous TTN pathogenic variant in a metatranscript-only exon: the c.36400A > T, p.

Dystrophin myonuclear domain restoration governs treatment efficacy in dystrophic muscle.

Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called "basal sarcolemmal dystrophin units (BSDUs).

Dlk1-Dio3 cluster miRNAs regulate mitochondrial functions in the dystrophic muscle in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by impaired expression of dystrophin. Whereas mitochondrial dysfunction is thought to play an important role in DMD, the mechanism of this dysfunction remains to be clarified. Here we demonstrate that in DMD and other muscular dystrophies, a large number of Dlk1-Dio3 clustered miRNAs (DD-miRNAs) are coordinately up-regulated in regenerating myofibers and in the serum.

Objective Evaluation of Clinical Actionability for Genes Involved in Myopathies: 63 Genes with a Medical Value for Patient Care.

The implementation of high-throughput diagnostic sequencing has led to the generation of large amounts of mutational data, making their interpretation more complex and responsible for long delays. It has been important to prioritize certain analyses, particularly those of "actionable" genes in diagnostic situations, involving specific treatment and/or management. In our project, we carried out an objective assessment of the clinical actionability of genes involved in myopathies, for which only few data obtained methodologically exist to date.

Deciphering the Molecular Mechanism of Incurable Muscle Disease by a Novel Method for the Interpretation of miRNA Dysregulation.

It is now well-established that microRNA dysregulation is a hallmark of human diseases, and that aberrant expression of miRNA is not randomly associated with human pathologies but plays a causal role in the pathological process. Investigations of the molecular mechanism that links miRNA dysregulation to pathophysiology can therefore further the understanding of human diseases. The biological effect of miRNA is thought to be mediated principally by miRNA target genes.

Skeletal Muscle Cells Derived from Induced Pluripotent Stem Cells: A Platform for Limb Girdle Muscular Dystrophies.

Limb girdle muscular dystrophies (LGMD), caused by mutations in 29 different genes, are the fourth most prevalent group of genetic muscle diseases. Although the link between LGMD and its genetic origins has been determined, LGMD still represent an unmet medical need. Here, we describe a platform for modeling LGMD based on the use of human induced pluripotent stem cells (hiPSC).

Origin and mobility of Iron Age Gaulish groups in present-day France revealed through archaeogenomics.

The Iron Age period occupies an important place in French history because the Gauls are regularly presented as the direct ancestors of the extant French population. We documented here the genomic diversity of Iron Age communities originating from six French regions. The 49 acquired genomes permitted us to highlight an absence of discontinuity between Bronze Age and Iron Age groups in France, lending support to a cultural transition linked to progressive local economic changes rather than to a massive influx of allochthone groups.

Co-Administration of Simvastatin Does Not Potentiate the Benefit of Gene Therapy in the mdx Mouse Model for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is the most common and cureless muscle pediatric genetic disease, which is caused by the lack or the drastically reduced expression of dystrophin. Experimental therapeutic approaches for DMD have been mainly focused in recent years on attempts to restore the expression of dystrophin. While significant progress was achieved, the therapeutic benefit of treated patients is still unsatisfactory.

Anoctamin 5 Knockout Mouse Model Recapitulates LGMD2L Muscle Pathology and Offers Insight Into in vivo Functional Deficits.

Mutations in the Anoctamin 5 (Ano5) gene that result in the lack of expression or function of ANO5 protein, cause Limb Girdle Muscular Dystrophy (LGMD) 2L/R12, and Miyoshi Muscular Dystrophy (MMD3). However, the dystrophic phenotype observed in patient muscles is not uniformly recapitulated by ANO5 knockout in animal models of LGMD2L. Here we describe the generation of a mouse model of LGMD2L generated by targeted out-of-frame deletion of the Ano5 gene.

A revised model for mitochondrial dysfunction in Duchenne muscular dystrophy.

We recently identified a signaling pathway that links the upregulation of miR-379 with a mitochondrial response in dystrophic muscle. In the present commentary, we explain the significance that this pathway may have in mitochondrial dysfunction in Duchenne muscular dystrophy (DMD). We identified the upregulation of miR-379 in the serum and muscles of DMD animal models and patients.

Titin M-line insertion sequence 7 is required for proper cardiac function in mice.

Titin is a giant sarcomeric protein that is involved in a large number of functions, with a primary role in skeletal and cardiac sarcomere organization and stiffness. The titin gene (TTN) is subject to various alternative splicing events, but in the region that is present at the M-line, the only exon that can be spliced out is Mex5, which encodes for the insertion sequence 7 (is7). Interestingly, in the heart, the majority of titin isoforms are Mex5+, suggesting a cardiac role for is7.