The lncRNA 44s2 Study Applicability to the Design of 45-55 Exon Skipping Therapeutic Strategy for DMD.

In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.

[Antisense oligonucleotides in SMA: lessons learned and literature data]. / Les oligonucléotides anti-sens dans la SMA - Retour d'expérience et données de la littérature.

TITLE: Les oligonucléotides anti-sens dans la SMA - Retour d'expérience et données de la littérature. ABSTRACT: Une révolution est actuellement en cours dans le domaine des maladies neuromusculaires avec l'arrivée de nouvelles thérapies. L'amyotrophie spinale (SMA ou spinal muscular atrophy) est parmi les maladies pionnières de ce bouleversement thérapeutique. Le premier traitement approuvé et mis sur le marché en Europe et aux États-Unis est un oligonucléotide antisens dénommé nusinersen et commercialisé par le laboratoire Biogen sous le nom de Spinraza®. Il a comme indication les SMA de types 1, 2 et 3. La première injection de Spinraza® dans le cadre d'une ATU/EAP (Autorisation Temporaire d'Utilisation/Expanded Access Program ou programmes d'accès étendu) a été réalisée en France par le centre d'essais l-Motion. Les résultats des essais cliniques et des données de la littérature sur l'utilisation du nusinersen dans la SMA infantile sont discutés dans cette revue. Ces études rapportent une amélioration de la fonction motrice chez les patients SMA tous types confondus y compris les patients de type 3 [1, 2]. Une administration précoce du traitement s'accompagne d'une meilleure réponse clinique. Une meilleure compréhension de l'hétérogénéité génétique et clinique devient indispensable dans le monitoring et le suivi à long terme de ces patients.

X-linked myotubular myopathy: A prospective international natural history study.

OBJECTIVES: Because X-linked myotubular myopathy (XLMTM) is a rare neuromuscular disease caused by mutations in the MTM1 gene with a large phenotypic heterogeneity, to ensure clinical trial readiness, it was mandatory to better quantify disease burden and determine best outcome measures. METHODS: We designed an international prospective and longitudinal natural history study in patients with XLMTM and assessed muscle strength and motor and respiratory functions over the first year of follow-up. The humoral immunity against adeno-associated virus serotype 8 was also monitored. RESULTS: Forty-five male patients aged 3.5 months to 56.8 years were enrolled between May 2014 and May 2017. Thirteen patients had a mild phenotype (no ventilation support), 7 had an intermediate phenotype (ventilation support less than 12 hours a day), and 25 had a severe phenotype (ventilation support 12 or more hours a day). Most strength and motor function assessments could be performed even in very weak patients. Motor Function Measure 32 total score, grip and pinch strengths, and forced vital capacity, forced expiratory volume in the first second of exhalation, and peak cough flow measures discriminated the 3 groups of patients. Disease history revealed motor milestone loss in several patients. Longitudinal data on 37 patients showed that the Motor Function Measure 32 total score significantly decreased by 2%. Of the 38 patients evaluated, anti-adeno-associated virus type 8 neutralizing activity was detected in 26% with 2 patients having an inhibitory titer >1:10. CONCLUSIONS: Our data confirm that XLMTM is slowly progressive for male survivors regardless of their phenotype and provide outcome validation and natural history data that can support clinical development in this population. CLINICALTRIALSGOV IDENTIFIER: NCT02057705.

Nusinersen in patients older than 7 months with spinal muscular atrophy type 1: A cohort study.

OBJECTIVE: To evaluate the safety and clinical efficacy of nusinersen in patients older than 7 months with spinal muscular atrophy type 1 (SMA1). METHODS: Patients with SMA1 were treated with nusinersen by intrathecal injections as a part of the Expanded Access Program (EAP; NCT02865109). We evaluated patients before treatment initiation (M0) and at 2 months (M2) and 6 months (M6) after treatment initiation. Survival, respiratory, and nutritional data were collected. Motor function was assessed with the modified Hammersmith Infant Neurologic Examination Part 2 (HINE-2) and physiotherapist scales adjusted to patient age (Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders and the Motor Function Measure 20 or 32). RESULTS: We treated 33 children ranging in age from 8.3 to 113.1 months between December 2016 and May 2017. All patients were alive and were continuing treatment at M6. Median progress on the modified HINE-2 score was 1.5 points after 6 months of treatment (p < 0.001). The need for respiratory support significantly increased over time. There were no statistically significant differences between patients presenting with 2 and those presenting with 3 copies of the survival motor neuron 2 (SMN2) gene. CONCLUSIONS: Our results are in line with the phase 3 study for nusinersen in patients with SMA1 treated before 7 months of age and indicate that patients benefit from nusinersen even at a later stage of the disease. CLINICALTRIALSGOV IDENTIFIER: NCT02865109. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that for patients with SMA1 who are older than 7 months, nusinersen is beneficial.

miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context.

BACKGROUND: Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the DMD gene coding for dystrophin, a protein being part of a large sarcolemmal protein scaffold that includes the neuronal nitric oxide synthase (nNOS). The nNOS was shown to play critical roles in a variety of muscle functions and alterations of its expression and location in dystrophic muscle fiber leads to an increase of the muscle fatigability. We previously revealed a decrease of nNOS expression in BMD patients all presenting a deletion of exons 45 to 55 in the DMD gene (BMDd45-55), impacting the nNOS binding site of dystrophin. Since several studies showed deregulation of microRNAs (miRNAs) in dystrophinopathies, we focused on miRNAs that could target nNOS in dystrophic context. METHODS: By a screening of 617 miRNAs in BMDd45-55 muscular biopsies using TLDA and an in silico study to determine which one could target nNOS, we selected four miRNAs. In order to select those that targeted a sequence of 3'UTR of NOS1, we performed luciferase gene reporter assay in HEK393T cells. Finally, expression of candidate miRNAs was modulated in control and DMD human myoblasts (DMDd45-52) to study their ability to target nNOS. RESULTS: TLDA assay and the in silico study allowed us to select four miRNAs overexpressed in muscle biopsies of BMDd45-55 compared to controls. Among them, only the overexpression of miR-31, miR-708, and miR-34c led to a decrease of luciferase activity in an NOS1-3'UTR-luciferase assay, confirming their interaction with the NOS1-3'UTR. The effect of these three miRNAs was investigated on control and DMDd45-52 myoblasts. First, we showed a decrease of nNOS expression when miR-708 or miR-34c were overexpressed in control myoblasts. We then confirmed that DMDd45-52 cells displayed an endogenous increased of miR-31, miR-708, and miR-34c and a decreased of nNOS expression, the same characteristics observed in BMDd45-55 biopsies. In DMDd45-52 cells, we demonstrated that the inhibition of miR-708 and miR-34c increased nNOS expression, confirming that both miRNAs can modulate nNOS expression in human myoblasts. CONCLUSION: These results strongly suggest that miR-708 and miR-34c, overexpressed in dystrophic context, are new actors involved in the regulation of nNOS expression in dystrophic muscle.

DMD and West syndrome.

Duchenne Muscular Dystrophy (DMD) is the most frequent muscular dystrophy in childhood, with a worldwide incidence of one in 5000 live male births. It is due to mutations in the dystrophin gene leading to absence of full-length dystrophin protein. Central nervous system involvement is well-known in Duchenne Muscular Dystrophy. The multiple dystrophin isoforms expressed in brain have important roles in cerebral development and functioning. The association of Duchenne Muscular Dystrophy with seizures has been reported, and there is a higher prevalence of epilepsy in Duchenne Muscular Dystrophy patients (between 6.3% and 12.3%) than in the general pediatric population (0.5-1%). Duchenne Muscular Dystrophy patients may present with focal seizures, generalized tonic-clonic seizures or absences. We report on two boys in whom Duchenne Muscular Dystrophy is associated with epileptic spasms and hypsarrhythmia that fulfil the criteria for West syndrome, thus extending the spectrum of seizure types described in Duchenne Muscular Dystrophy patients.

Mild clinical presentation in KLHL40-related nemaline myopathy (NEM 8).

Nemaline myopathies are clinically and genetically heterogeneous muscle diseases characterized by the presence of nemaline bodies (rods) in muscle fibers. Mutations in the KLHL40 (kelch-like family member 40) gene (NEM 8) are common cause of severe/lethal nemaline myopathy. We report an 8-year-old girl born to consanguineous Moroccan parents, who presented with hypotonia and poor sucking at birth, delayed motor development, and further mild difficulties in walking and fatigability. A muscle biopsy revealed the presence of nemaline bodies. KLHL40 gene Sanger sequencing disclosed a never before reported pathogenic homozygous mutation which resulted in absent KLHL40 protein expression in the muscle. This further expands the phenotypical spectrum of KLHL40 related nemaline myopathy.