Centronuclear myopathies (CNM) are rare congenital myopathies characterized by abnormal centralization of nuclei in muscle fibers. Three main forms of CNM are distinguished: The X-linked recessive CNM (also called X-linked myotubular myopathy, XLMTM) due to mutations in myotubularin (MTM1), the autosomal recessive CNM (AR-CNM) due to mutations in amphiphysin 2 (BIN1), and the autosomal dominant CNM (AD-CNM) due to mutations in dynamin 2 (DNM2). There is no therapies available for patients affected by CNMs. The teams of Marc Bitoun (UMRS974, Myology Institute, Paris) and Belinda Cowling/Jocelyn Laporte (IGBMC, Illkirch) have been involved for many years in the identification and the functional consequences of mutations in CNMs, in the development of animal models and therapeutic strategies for the CNMs. During the last years, common efforts of the two teams supported by the “DynaMuscle” ANR program led to essential breakthrough for the development of innovative therapeutic strategies based on modulation of the DNM2 expression in two forms of CNM. First, proof of concept for allele-specific silencing therapy was achieved in AD-CNM by targeting the most frequent DNM2 mutation in the Knock-in-Dnm2R465W mouse model (KI-Dnm2) and in patient-derived cells. Second, cross-therapy by reduction of DNM2 was established as efficient therapeutic strategy for XLMTM in which DNM2 is over-expressed. DynaTher aims at “converting the try” by pursuing preclinical developments for these two “DNM2 therapies” by tackling the following questions: - What preclinical developments are required for allele-specific therapy for the p.R645W DNM2 mutation in AD-CNM? We will investigate the long-term maintenance of efficacy and benefit of systemic treatment in young KI-Dnm2 mice. We will also optimize the therapeutic benefit in old mice. - Can allele-specific silencing be extended to other AD-CNM DNM2 mutations and is it possible to develop “pan-mutations” tools to avoid a mutation-based strategy? We will screen for allele-specific siRNA able to silence 5 other DNM2 mutations in patient-derived cells. We will also develop “pan-mutations” allele-specific-siRNA against the two versions of frequent heterozygous non-pathological single nucleotide polymorphisms present in the DNM2 mRNA. - Is the cross-therapy strategy also efficient for other forms of CNM, thus increasing the number of patients that can be treated by this approach? Based on our previous proof of principle for XLMTM, we will extend this therapy in mouse models of AR-CNM and AD-CNM. - Do siRNAs against DNM2 have “off-target effects”? That will be answer by associating transcriptome and proteome analyses in human cells from patients affected by the three main forms of CNM allowing also to uncover common and specific altered pathways in CNMs and readouts for future clinical trials. - Can DNM2 therapy be improved by targeting specific DNM2 isoform or by other delivery methods and be extended to other neuromuscular disorders? Therapeutic benefit of reduction of the DNM2 muscle-specific isoform will be determined in Knock-out-Mtm1 and KI-Dnm2 mouse models. Non-viral delivery methods will be also developed. Finally, we will identify novel applications for DNM2 therapy through a screening for elevated DNM2 expression in a panel of neuromuscular disorders. The ambition of DynaTher is to accelerate the preclinical development of these approaches to ultimately provide the most efficient and safe molecular tools targeting DNM2 required for the first gene therapy clinical trial for autosomal CNMs.

Centronuclear myopathies (CNM) are rare congenital myopathies characterized by abnormal central position of nuclei in the muscle fibers in absence of muscle regeneration. The three main forms include the X-linked recessive CNM due to Myotubularin (MTM1) mutations, and the autosomal forms due to dominant mutations of Dynamin 2 and recessive mutations of Amphiphysin 2 (BIN1) and Ryanodine receptor 1. Dynamin 2 (DNM2) is a key actor of membrane trafficking and a regulator of both actin and microtubule cytoskeletons. Our two teams developed potential therapies for CNMs based on reduction of the DNM2 expression. The first therapy is based on RNA interference-mediated allele-specific silencing of mutated or non-mutated DNM2 mRNAs. We developed siRNA against the most frequent DNM2 mutation causing the dominant CNM and versatile siRNA able to silence all the DNM2 mutations. Their therapeutic benefit was validated in one animal model and patient-derived cells. The second therapy, devoted to reduce overall DNM2 expression especially using antisense oligonucleotides, successfully rescued animal models of the MTM1-, BIN1-, -and DNM2-linked CNM genetic forms. Based on these first proof of concepts, DynANR aims at proceeding to preclinical development of these two therapies for CNMs and extend their field of application through 4 tasks: 1. Development of the allele-specific therapy. We will optimize the Adeno-Associated Virus as vector for this therapy in a mouse model of classical adult form of dominant CNM and assess therapeutic efficacy of allele-specific silencing in muscle cells from patients and in a second mouse model mimicking the most severe form linked to DNM2 mutations. The benefit of the versatile allele-specific siRNA will be also assessed in MTM1- and BIN1-linked CNMs. 2. Development of the pan-allelic therapy. We will assess the therapeutic benefit of DNM2 overall reduction in another genetic form of CNM. We will also develop a new generation of oligonucleotides using another chemistry and another mode of action. 3. Gene editing. We will assess the efficacy of gene editing by CRISPR/Cas9 as a new technology for reducing DNM2 expression in both allele-specific and pan-allelic approaches. The therapeutic benefit will be assessed in mouse models of the MTM1- and DNM2-linked CNMs. 4. Biomarkers and new application. We will determine biomarkers to follow disease progression and therapy efficiency from transcriptomic profiling of CNM patient muscles. We will also determine the DNM2 level in a panel of neuromuscular disorders to highlight novel indications for DNM2 reduction. By a reinforced interaction between our two teams, the ambition of DynANR is to accelerate the preclinical development of DNM2 therapies required for clinical trials targeting several forms of CNMs. The four tasks are based on preliminary data and on expertise, concepts, and methodologies already developed by the 2 partners, especially through previous ANR supports.

Intracellular organization and transport is fundamental for the function of eukaryotic cells. Dynamins are a prototype class of mechano-enzymes and key regulators of membrane fission. These large GTPases regulate the scission of nascent vesicles from the plasma membrane during endocytosis; they also participate in the formation and trafficking of vesicles from intracellular compartments, and fusion processes. Moreover, dynamins bind to both microtubules and actin and potentially regulate these cytoskeletons. While tremendous efforts have been made to understand dynamin-dependent membrane fission, the cellular function of dynamins in specific tissues and their physiological role remain poorly understood, especially for the ubiquitous member of the family, dynamin 2. Describing the physiological role of dynamin 2 (DNM2) is of particular importance as DNM2 mutations cause two unrelated neuromuscular disorders for which no treatments are available and pathomechanisms are largely unknown: dominant Charcot-Marie-Tooth neuropathies and dominant centronuclear myopathy. Centronuclear myopathies (CNM) are rare neuromuscular disorders characterized by muscle weakness and intracellular disorganization of muscle fibres. A crucial role for DNM2 in muscle homeostasis was supported by our studies in mice, however data from cell culture showed these mutations impact on only some membrane trafficking routes controlled by dynamins, suggesting that specific functions of DNM2 are important in normal and diseased muscle, and that additional functions remain to be discovered in a tissue-specific context. Moreover, we have shown DNM2 represents a therapeutic target for several forms of CNM. Marc Bitoun (Myology Institute, Paris) and Jocelyn Laporte (IGBMC, Illkirch)’s teams have been involved for many years in the identification of the functional consequences of mutations in DNM2 and in the development of animal models for different forms of centronuclear myopathies. It is now crucial to define the physiological role of DNM2 in muscle, and this will be instrumental to understand the pathomechanisms linked to DNM2 mutations and develop future therapies for patients. We will thus synergize to decipher the roles of dynamin 2 in skeletal muscle under normal and pathological conditions, and will tackle the following relevant questions: - what is the basis for the muscle–specific function of the ubiquitously expressed DNM2 ? (Task 1). We will identify and validate tissue-specific isoforms and binding partners. - what is the impact of DNM2 alteration on its muscle-specific functions ? (Task 2). We will study the impact of patient mutations on localization and binding partners, and characterize novel mouse models for DNM2-loss in muscle to decipher its physiological importance. - what are the cellular pathways controlled by DNM2 in muscle ? (Task 3). We will especially investigate costamere formation and maintenance, mitochondria and triads that are key structures for muscle metabolism and contraction, and autophagy flux. This will also lead to a better understanding of CNM pathomechanisms. - what are the best pre-clinical approaches to rescue DNM2-related myopathy ? (Task 4). Based on preliminary data and a better understanding of the role of DNM2 in muscle, we will test genetic modulation, allele specific inhibition and autophagy modulators to improve and revert the CNM phenotypes. By sharing tools and expertise available in our 2 teams, we expect to achieve important steps toward preclinical development of therapies for DNM2-related CNM. The two partners bring together a set of unique and complementary knowledge, expertise, models and tools, and have access to patient samples for validation. Overall, this multidisciplinary project aims to detail for the first time the poorly studied physiological roles of dynamin in muscle under both normal and disease conditions, to ultimately provide novel and innovative therapeutic strategies leading to clinical trials.

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults, characterised by myotonia, muscle weakness and progressive muscle atrophy. DM1 is caused by the expression of mutated RNAs containing expansions of CUG repeats (CUGexp-RNA) that form nuclear aggregates and alter the activity of RNA-binding proteins and the metabolism of certain RNAs. Although a number of muscular symptoms such as myotonia have been associated with abnormalities in the regulation of alternative splicing of target transcripts, the mechanisms involved in the dystrophic process are still poorly understood and animal models of DM1 do not reproduce, or only partially reproduce, this most disabling phenotype of the disease. Using new mouse models enabling cell/tissue-specific expression of the DM1 mutation in adults, we will study muscle changes following specific expression of CUGexp-RNA in muscle fibres and/or in muscle stem cells in order to determine their respective contribution to the dystrophic process that progressively develops in DM1 patients. Physiological and histological analyses of the muscles will be supplemented by transcriptomic analyses using RNAseq and also at the level of single nuclei using snRNAseq in order to identify and characterize the molecular alterations induced by the expression of CUGexp-RNA that will affect their cellular fate. Spatial transcriptomic or RNAscope analyses will complement these analyses in order have a comprehension view of the cellular mechanisms involved in the degenerative process found in the skeletal muscle of DM1 patients.

The SEDUCE proposal aims at establishing an innovative approach to identify new therapeutic molecules for alternate-splicing related diseases and to develop new screening tools for drug discovery approaches driven by academia and industry. Pre-mRNA splicing is a fundamental process in mammalian gene expression and alternative RNA splicing plays a considerable role in generating protein diversity. During this process, particular exons of a gene will be included within or excluded from the final maturated mRNA, and the resulting transcripts generate diverse protein isoforms. Alternative splicing has been proposed as one of the major mechanisms contributing to protein diversity. Increasing evidence has shown that the disruption of alternative splicing negatively impacts health and contributes to human diseases, including cancer, diabetes, and neuromuscular diseases. The recent realization that up to 50% of genetic diseases involve splicing mutations has driven the development of several therapeutic approaches to correct aberrant splicing. Among these, the identification of small molecules capable to modulate splicing has been accelerated in the last decade with the technical development of large-scale cell-based screens. However, up to date, the success rate for identifying a splicing modulator that reaches the market is extremely low. Several parameters can explain this failure 1). the use of reporter constructs that contain only a restricted part of the gene of interest 2). the use of transformed cells instead of disease-relevant cell types. Therefore, one of the most important challenges for the future development of small molecule modulator will depend upon how well the specificity of the effects can be optimized. Altogether, these bottlenecks largely block the deployment of drug discovery campaigns and therefore abrogate the development of new medicines curing alternate splicing related diseases early on in the drug development process. In this context, SEDUCE consortium proposes to combine the use of disease-specific human stem cells differentiated into relevant cell types with high throughput RT-qPCR screening to identify new therapeutics for two distinct alternative splicing related neuromuscular diseases: spinal muscular atrophy and myotonic dystrophy type 1. Overall, our proposal has several objectives (1) identify and optimize new splice modulators for SMA and DM1, (2) decipher their mechanisms of action (3) validate their action in vivo and (4) convert these new molecules into marketable products. Ultimately, our aim is to deliver a meaningful technology that will accelerate the development of therapeutics for the growing list of diseases in which the process of pre-mRNA splicing is altered while ensuring its direct availability to academic lab or pharmaceuticals companies for screening campaigns. To achieve these goals, the consortium has secured all the necessary expertise : Partner 1 (Cécile Martinat, I-Stem) was one of the first laboratory developing new cellular models based on disease-specific human pluripotent stem cells such as DM1 and SMA. Partner 2 (Eric Perret, Evotec) brings an industrial expertise in the development of successful RT-qPCR HTS based, in the access and optimization of chemical librairies and the expertise of hit optimization. Partner 3 and Partner 4 (Denis Furling, Institute of Myology and Nicolas Charlet, IGBMC) are leading experts in deciphering molecular mechanisms involved in normal and pathological alternate splicing as well as in testing splicing and phenotype correction in vivo in mouse models. Our proposal offers a genuinely innovative opportunity to push beyond the limitations of current models and promises to open up major new “assay development space” by increasing our understanding of the regulation of alternate splicing, identifying disease specific splicing modulators and offering a platform that can be applied to a wide range of research areas.