Severe PATCHED1 deficiency in cancer-prone Gorlin patient cells results in intrinsic radiosensitivity

PURPOSE: Gorlin syndrome (or basal-cell nevus syndrome) is a cancer-prone genetic disease in which hypersusceptibility to secondary cancer and tissue reaction after radiation therapy is debated, as is increased radiosensitivity at cellular level. Gorlin syndrome results from heterozygous mutations in the PTCH1 gene for 60% of patients, and we therefore aimed to highlight correlations between intrinsic radiosensitivity and PTCH1 gene expression in fibroblasts from adult patients with Gorlin syndrome. METHODS AND MATERIALS: The radiosensitivity of fibroblasts from 6 patients with Gorlin syndrome was determined by cell-survival assay after high (0.5-3.5 Gy) and low (50-250 mGy) γ-ray doses. PTCH1 and DNA damage response gene expression was characterized by real-time polymerase chain reaction and Western blotting. DNA damage and repair were investigated by γH2AX and 53BP1 foci assay. PTCH1 knockdown was performed in cells from healthy donors by using stable RNA interference. Gorlin cells were genotyped by 2 complementary sequencing methods. RESULTS: Only cells from patients with Gorlin syndrome who presented severe deficiency in PATCHED1 protein exhibited a significant increase in cellular radiosensitivity, affecting cell responses to both high and low radiation doses. For 2 of the radiosensitive cell strains, heterozygous mutations in the 5' end of PTCH1 gene explain PATCHED1 protein deficiency. In all sensitive cells, DNA damage response pathways (ATM, CHK2, and P53 levels and activation by phosphorylation) were deregulated after irradiation, whereas DSB repair recognition was unimpaired. Furthermore, normal cells with RNA interference-mediated PTCH1 deficiency showed reduced survival after irradiation, directly linking this gene to high- and low-dose radiosensitivity. CONCLUSIONS: In the present study, we show an inverse correlation between PTCH1 expression level and cellular radiosensitivity, suggesting an explanation for the conflicting results previously reported for Gorlin syndrome and possibly providing a basis for prognostic screens for radiosensitive patients with Gorlin syndrome and PTCH1 mutations

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

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

Restauration de la dystrophine par saut d'exons chez le modèle canin GRMD ; Augmentation de la masse musculaire par inhibition de la myostatine (rationnel thérapeutique pour DMD ?)

La dystrophie musculaire de Duchenne (DMD) est une maladie progressive et sévère qui demeure incurable malgré le développement de différentes stratégies thérapeutiques. Nous avons décidé de travailler sur deux approches innovantes. Notre premier objectif était de mettre au point des outils moléculaires pour une correction post-transcriptionnelle du gène de la dystrophine. La technique du saut d'exon permet de restaurer le cadre de lecture dans le cas de nombreuses mutations et d'obtenir une protéine plus courte mais fonctionnelle. Nous avons obtenu un saut d'exon(s) efficace et stable après une seule administration du vecteur AAV exprimant les séquences anti-sens liées à un petit ARN nucléaire U7. Nos résultats montrent une restauration de la dystrophine fonctionnelle chez le modèle canin GRMD et localement la correction de la dystrophie musculaire. Notre second objectif était d'améliorer le phénotype DMD en augmentant la masse musculaire grâce à la sous-expression de la myostatine et de vérifier une possible amélioration de la régénération musculaire. Nous avons démontré que le propeptide de la myostatine était un agent efficace pour l'accroissement de la masse musculaire et le bénéfice fonctionnel reste à établir dans les modèles dystrophiques murin et canin.Duchenne Muscular Dystrophy (DMD) is a progressive devastating disorder that remains incurable in spite of development of different therapeutic strategies. We decided to work on two original approaches. Our first aim was to setup molecular tools for post-transcriptional correction by targeting exon skipping of frequent out-of-frame deletions in the dystrophin gene and obtain a shorter but functional protein. We have achieved persistent exon skipping by a single administration of an AAV vector expressing antisense sequences linked to a modified U7 small nuclear RNA. Our results show the sustained production of functional dystrophin at physiological levels in injected muscles of GRMD dog model and the correction of the muscular dystrophy. Our second aim was to improve the DMD phenotype by increasing the skeletal muscle mass thanks to the down-regulation of myostatin and verify a possible improvement of the muscular regeneration. We have demonstrated that the propeptide of myostatin is an effective agent for increasing muscle mass and the functional benefit continue to be established in dystrophic mouse and dog models.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

État actuel des connaissance sur l'utilisation des oligonucléotides antisense dans le traitement des maladie neuromusculaire

International audienceNeuromuscular disorders include a wide range of diseases affecting the peripheral nervous system, which are primarily characterized by progressive muscle weakness and wasting. While there were no effective therapies until recently, several therapeutic approaches have advanced to clinical trials in the past few years. Among these, the antisense technology aiming at modifying RNA processing and function has remarkably progressed and a few antisense oligonucleotides (ASOs) have now been approved. Despite these recent clinical successes, several ASOs have also failed and clinical programs have been suspended, in most cases when the route of administration was systemic, highlighting the existing challenges notably with respect to effective ASO delivery. In this review we summarize the recent advances and current status of antisense based-therapies for neuromuscular disorders, using successful as well as unsuccessful examples to highlight the variability of outcomes depending on the target tissue and route of administration. We describe the different ASO-mediated therapeutic approaches, including splice-switching applications, steric-blocking strategies and targeted gene knock-down mediated by ribonuclease H recruitment. In this overview, we discuss the merits and challenges of the current ASO technology, and discuss the future of ASO development

Pre-clinical dose-escalation studies establish a therapeutic range for U7snRNA-mediated DMD exon 2 skipping

Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein that typically results from truncating mutations in the DMD gene. Current exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) by skipping an adjacent exon, in order to restore an ORF that allows translation of an internally deleted yet partially functional protein, as is seen with many patients with the milder Becker muscular dystrophy (BMD) phenotype. In contrast to that approach, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild-type protein. We have developed an adeno-associated virus (AAV)-based U7snRNA exon-skipping approach directed toward exon 2, duplications of which represent 10% of all DMD duplication mutations. Deletion of exon 2 results in utilization of an exon 5 internal ribosome entry site (IRES) that allows translation beginning in exon 6 of a highly protective dystrophin protein, providing a wide therapeutic window for treatment. Both intramuscular and systemic administration of this vector in the Dup2 mouse model results in robust dystrophin expression and correction of muscle physiologic defects, allowing dose escalation to establish a putative minimal efficacious dose for a human clinical trial