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The nucleic acid therapeutics field has made tremendous progress in the past decades. Continuous advances in chemistry and design have led to many successful clinical applications, eliciting even more interest from researchers including both academic groups and drug development companies. Many preclinical studies in the field focus on improving the delivery of antisense oligonucleotide drugs (ONDs) and/or assessing their efficacy in target tissues, often neglecting the evaluation of toxicity, at least in early phases of development. A series of consensus recommendations regarding regulatory considerations and expectations have been generated by the Oligonucleotide Safety Working Group and the Japanese Research Working Group for the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S6 and Related Issues (WGS6) in several white papers. However, safety aspects should also be kept in sight in earlier phases while screening and designing OND to avoid subsequent failure in the development phase. Experts and members of the network "DARTER," a COST Action funded by the Cooperation in Science and Technology of the EU, have utilized their collective experience working with OND, as well as their insights into OND-mediated toxicities, to generate a series of consensus recommendations to assess OND toxicity in early stages of preclinical research. In the past few years, several publications have described predictive assays, which can be used to assess OND-mediated toxicity

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 ( Dmd EGFP ), 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).” These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber—with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle–tendon junctions.

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare vascular disorder causing abnormal vessel formation and characterized by autosomal dominant transmission. The associated considerable variability in symptoms and clinical severity complicate the management of the disease. In clinical routine, 3 main genes,ACVRL1(also known asALK1),ENGandSMAD4are screened for pathogenic variants at the origin of HHT. About 80% of HHT cases are caused by pathogenic coding variants inACVRL1andENG. However, at least 15% remain with no molecular explanations in the 3 main genes. We here report the identification of 2 never reported variants, c.-79C>T and c.-68G>A, in the 5’UTR ofENGin 2 unrelated HHT patients. These 2 variants are predicted to create upstream AUGs (uAUGs) in the 5’UTR, which are in frame with a stop codon located within the CoDing Sequence (CDS), thus generating Overlapping upstream Open reading frames (uoORFs). In other cases, uAUGs can lead to fully upstream ORFs (uORFs) when associated with stop codons located within the 5’UTR or to elongated CDS (eCDS) when in frame with the CDS and associated with the main stop codon.In order to assess the pathogenicity of theseENGvariants, we performedin vitrofunctional assays based on the expression of wild-type and mutant constructs in human cells. We found that these 5’UTRENGvariants were associated with a decrease of protein levels in HeLa and HUVEC cells. They were also associated with a decreased ability to activate BMP9-stimulated ALK1 receptor in a BMP-response element (BRE) assay. This assay is a mandatory element before providing a definitive molecular diagnosis and has been so far applied only on codingENGvariants. We applied the same experimental workflow on 3 additional uoORF-creating variants (c.-142A>T, c.-127C>T and c.-10C>T) and one eCDS-creating variant (c.-9G>A) in the 5’UTR ofENGpreviously reported in HHT patients. We found that all the analysed uoORF-creating variants alter endoglin levels and function. A comparison of our experimental results with patients’ clinical characteristics suggests that uoORF-creating variants leading to ENG protein levels ≤ 40%in vitrowould be associated with HHT. Additional experiments relying on an artificial deletion in our mutated constructs show that created uAUGs predicted to create uoORFs are able to initiate the translation indicating that the associated effect is likely caused by an alteration of the translation mechanism.Overall, we here identified two never reported 5’UTRENGvariations in HHT patients and shed new lights on the role of upstream ORFs on ENG regulation. Our findings contribute to the amelioration of molecular diagnosis in HHT.

Les avancées en recherches génétique et génomique ne cessent d’accroître nos connaissances des maladies héréditaires. Un nombre croissant de ces maladies relève d’épissages aberrants qui représentent des cibles idéales pour les approches correctives centrées sur l’ARN. De nouvelles stratégies, en particulier médicamenteuses, visant à exclure ou à ré-inclure des exons lors du processus d’épissage, ont ainsi émergé et plusieurs molécules ont récemment obtenu des autorisations de mise sur le marché, notamment pour le traitement de la dystrophie musculaire de Duchenne et de l’amyotrophie spinale, suscitant de plus en plus d’intérêt et d’espoir. Parmi ces molécules, les oligonucléotides antisens, ou ASO, ont connu un réel essor et font l’objet de progrès constants en matière de modifications chimiques et de conception. Toutefois, leur biodistribution après administration par voie générale demeure souvent limitée, et le développement de chimies alternatives plus performantes et de nouveaux systèmes d’adressage est devenu un axe de recherche très actif. En parallèle, l’utilisation de petites molécules présentant une excellente biodistribution, ou de vecteurs viraux pour véhiculer les séquences antisens, est également explorée. Dans cette Synthèse, nous présentons les dernières avancées de ces approches de modulation d’épissage à travers deux exemples de maladies neuromusculaires. Nous discutons de leurs avantages et des principales limitations actuelles.

International audience; Objectives: Duchenne muscular dystrophy is associated with various degrees of cognitive impairment and behavioral disturbances. Emotional and memory deficits also constitute reliable outcome measures to assess efficacy of treatments in the mdx mouse lacking the muscle and neuronal full-length dystrophins. The present study aimed to evaluate whether these deficits could be alleviated by the restoration of brain dystrophin.Methods: We performed intracerebroventricular administration of a new potent tricyclo-DNA antisense oligonucleotide (tcDNA-ASO) containing a full phosphodiester backbone conjugated to a palmitic acid moiety (tcDNA-ASO), designed to skip the mutated exon 23 of mdx mice.Results: We first show that the tcDNA-ASO rescues expression of brain dystrophin to 10-30% of wild-type levels and significantly reduces the abnormal unconditioned fear responses in mdx mice in a dose-dependent manner, 5 weeks post-injection. Exon skipping efficiency, ASO biodistribution, protein restoration and effect on the fear response were optimal with a dose of 400 μg at 6-7 weeks post-injection, with synaptic-like expression in brain tissues such as the hippocampus and amygdala. Furthermore, this dose of tcDNA-ASO restored long-term memory retention of mdx mice in an object recognition task, but only had minor effects on fear conditioning.Interpretation: These results suggest for the first time that postnatal re-expression of brain dystrophin could reverse or at least alleviate some cognitive deficits associated with Duchenne muscular dystrophy. ANN NEUROL 2022;92:213-229.

The exon-52-deleted mdx52 mouse is a critical model of Duchenne muscular dystrophy (DMD), as it features a deletion in a hotspot region of the DMD gene, frequently mutated in patients. Deletion of exon 52 impedes expression of several brain dystrophins (Dp427, Dp260 and Dp140), thus providing a key model for studying the cognitive impairment associated with DMD and testing rescuing strategies. Here, using in vivo magnetic resonance imaging and neurohistology, we found no gross brain abnormalities in mdx52 mice, suggesting that the neural dysfunctions in this model are likely at the level of brain cellular functionalities. Then, we investigated emotional behavior and fear learning performance of mdx52 mice compared to mdx mice that only lack Dp427 to focus on behavioral phenotypes that could be used in future comparative preclinical studies. mdx52 mice displayed enhanced anxiety and a severe impairment in learning an amygdala-dependent Pavlovian association. These replicable behavioral outcome measures are reminiscent of the internalizing problems reported in a quarter of DMD patients, and will be useful for preclinical estimation of the efficacy of treatments targeting brain dysfunctions in DMD., Summary: The mdx52 mouse model of Duchenne muscular dystrophy lacks brain Dp427 and Dp140 dystrophins, and exhibits emotional, behavioral and learning phenotypes that are relevant for use in preclinical studies of the disorder.

Advances in genetic and genomic research continue to increase our knowledge of hereditary diseases, and an increasing number of them are being attributed to aberrant splicing, thus representing ideal targets for RNA modulation therapies. New strategies to skip or re-include exons during the splicing process have emerged and are now widely evaluated in the clinic. Several drugs have recently been approved in particular for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy. Among these molecules, antisense oligonucleotides, or ASOs, have gained increasing interest and have constantly been improved over the years through chemical modifications and design. However, their limited biodistribution following systemic administration still represents a major hurdle and the development of more potent alternative chemistries or new delivery systems has become a very active line of research in the past few years. In parallel, the use of small molecules with excellent biodistribution properties or of viral vectors to convey antisense sequences is also being investigated. In this review, we summarize the recent advances in splicing therapies through two examples of neuromuscular diseases and we discuss their main benefits and current limitations.Les approches thérapeutiques de modulation de l’épissage - Avancées et perspectives.Les avancées en recherches génétique et génomique ne cessent d’accroître nos connaissances des maladies héréditaires. Un nombre croissant de ces maladies relève d’épissages aberrants qui représentent des cibles idéales pour les approches correctives centrées sur l’ARN. De nouvelles stratégies, en particulier médicamenteuses, visant à exclure ou à ré-inclure des exons lors du processus d’épissage, ont ainsi émergé et plusieurs molécules ont récemment obtenu des autorisations de mise sur le marché, notamment pour le traitement de la dystrophie musculaire de Duchenne et de l’amyotrophie spinale, suscitant de plus en plus d’intérêt et d’espoir. Parmi ces molécules, les oligonucléotides antisens, ou ASO, ont connu un réel essor et font l’objet de progrès constants en matière de modifications chimiques et de conception. Toutefois, leur biodistribution après administration par voie générale demeure souvent limitée, et le développement de chimies alternatives plus performantes et de nouveaux systèmes d’adressage est devenu un axe de recherche très actif. En parallèle, l’utilisation de petites molécules présentant une excellente biodistribution, ou de vecteurs viraux pour véhiculer les séquences antisens, est également explorée. Dans cette Synthèse, nous présentons les dernières avancées de ces approches de modulation d’épissage à travers deux exemples de maladies neuromusculaires. Nous discutons de leurs avantages et des principales limitations actuelles.

International audience; The mdx52 mouse model of Duchenne muscular dystrophy (DMD) is lacking exon 52 of the DMD gene that is located in a hotspot mutation region causing cognitive deficits and retinal anomalies in DMD patients. This deletion leads to the loss of the dystrophin proteins, Dp427, Dp260 and Dp140, while Dp71 is preserved. The flash electroretinogram (ERG) in mdx52 mice was previously characterized by delayed dark-adapted b-waves. A detailed description of functional ERG changes and visual performances in mdx52 mice is, however, lacking. Here an extensive full-field ERG repertoire was applied in mdx52 mice and WT littermates to analyze retinal physiology in scotopic, mesopic and photopic conditions in response to flash, sawtooth and/or sinusoidal stimuli. Behavioral contrast sensitivity was assessed using quantitative optomotor response (OMR) to sinusoidally modulated luminance gratings at 100% or 50% contrast. The mdx52 mice exhibited reduced amplitudes and delayed implicit times in dark-adapted ERG flash responses, particularly in their b-wave and oscillatory potentials, and diminished amplitudes of light-adapted flash ERGs. ERG responses to sawtooth stimuli were also diminished and delayed for both mesopic and photopic conditions in mdx52 mice and the first harmonic amplitudes to photopic sine-wave stimuli were smaller at all temporal frequencies. OMR indices were comparable between genotypes at 100% contrast but significantly reduced in mdx52 mice at 50% contrast. The complex ERG alterations and disturbed contrast vision in mdx52 mice include features observed in DMD patients and suggest altered photoreceptor-to-bipolar cell transmission possibly affecting contrast sensitivity. The mdx52 mouse is a relevant model to appraise the roles of retinal dystrophins and for preclinical studies related to DMD.

Nucleic acid‐based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid‐based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics., Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics.

Progress with antisense oligonucleotide therapies opens a path for future development

International audience; Background: Dmdmdx, harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies. Methods: We generated a DmdEGFP-mdx reporter allele carrying in cis the mdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 23 excision. Results: Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx-23 mutation. Conclusion: Intravital imaging of DmdEGFP-mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.

International audience; Tricyclo-DNA (tcDNA) antisense oligonucleotides (ASOs) hold promise for therapeutic splice-switching applications and the treatment of Duchenne muscular dystrophy (DMD) in particular. We have previously reported the therapeutic potential of tcDNA-ASO in mouse models of DMD, highlighting their unique pharmaceutical properties and unprecedented uptake in many tissues after systemic delivery, including the heart and central nervous system. Following these encouraging results, we developed phosphorothioate (PS)-modified tcDNA-ASOs targeting the human dystrophin exon 51 (H51). Preliminary evaluation of H51 PS-tcDNA in mice resulted in unexpected acute toxicity following intravenous administration of the selected candidate. In vivo and in vitro assays revealed complement activation, prolonged coagulation times, and platelet activation, correlating with the observed toxicity. In this study, we identify a novel PS-tcDNA sequence-specific toxicity induced by the formation of homodimer-like structures and investigate the therapeutic potential of a detoxified PS-tcDNA targeting exon 51. Modification of the H51-PS-tcDNA sequence, while maintaining target specificity through wobble pairing, abolished the observed toxicity by preventing homodimer formation. The resulting detoxified wobble-tcDNA candidate did not affect coagulation or complement pathways any longer nor activated platelets in vitro and was well tolerated in vivo in mice, confirming the possibility to detoxify specific tcDNA-ASO candidates successfully.

International audience; Neuromuscular 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.

Loss-of-function mutations in the human coagulation factor 9 (F9) gene lead to hemophilia B. Here, we dissected the consequences and the pathomechanism of a non-coding mutation (c.2545A>G) in the F9 3’ untranslated region. Using wild type and mutant factor IX (FIX) minigenes we revealed that the mutation leads to reduced F9 mRNA and FIX protein levels and to lower coagulation activity of cell culture supernatants. The phenotype could not be compensated by increased transcription. The pathomechanism comprises the de novo creation of a binding site for the spliceosomal component U1snRNP, which is able to suppress the nearby F9 poly(A) site. This second, splicing-independent function of U1snRNP was discovered previously and blockade of U1snRNP restored mutant F9 mRNA expression. In addition, we explored the vice versa approach and masked the mutation by antisense oligonucleotides resulting in significantly increased F9 mRNA expression and coagulation activity. This treatment may transform the moderate/severe hemophilia B into a mild or subclinical form in the patients. This antisense based strategy is applicable to other mutations in untranslated regions creating deleterious binding sites for cellular proteins., Author summary The elucidation of the pathomechanisms of non-coding variants yields important insights into diseases as well as cellular processes causing the defect. Although these variants may account for the majority of phenotypic variation, only a minority of them can be explained mechanistically. The human coagulation factor 9 3’ UTR variant described here converts a non-essential sequence motif into a U1snRNP-binding site with deleterious effects on RNA 3’ end processing at the nearby poly(A) site. Poly(A) site suppression by U1snRNP was described before and it normally protects cellular mRNAs from premature termination. However, if misled by creation of a U1 site close the authentic poly(A) site as in the F9 3’ UTR, this nuclear surveillance mechanism results in the opposite. Since recognition by U1snRNP depends on sequence complementarity we were able to use antisense oligonucleotides to mask the mutant site and partially restored F9 mRNA levels. This antisense based strategy may be applicable to other variants in untranslated regions, which create deleterious binding sites for cellular proteins.

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder characterized by progressive muscle wasting that has currently no cure. Exon-skipping strategy represents one of the most promising therapeutic approaches that aim to restore expression of a shorter but functional dystrophin protein. The antisense field has remarkably progress over the last years with recent accelerated approval of the first antisense oligonucleotide-based therapy for DMD, Exondys 51, though the therapeutic benefit remains to be proved in patients. Despite clinical advances, the poor effective delivery to target all muscle remains the main hurdle for antisense drug therapy. This review describes the antisense-based exon-skipping approach for DMD, from proof-of-concept to first marketed drug. We discuss the main obstacles to achieve a successful exon-skipping therapy and the latest advances of the international community to develop more powerful chemistries and more sophisticated delivery systems in order to increase potency, bioavailability and safety. Finally, we highlight the importance of collaborative efforts and early dialogue between drug developers and regulatory agencies in order to overcome difficulties, find appropriate outcome markers and collect useful data.

Huntington's disease is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of huntingtin gene (HTT) encoding for a toxic polyglutamine protein. This disease is characterized by motor, psychiatric, and cognitive impairments. Currently, there is no disease modifying treatment. However, reducing the expression of the huntingtin protein (HTT) using antisense oligonucleotides (ASOs) has been shown as a promising therapeutic strategy. In this study, we explore the therapeutic potential of ASO made of tricyclo-DNA (tcDNA), a conformationally constrained DNA analog, to silence HTT. We used a gapmer ASO, containing central DNA nucleotides flanked by tcDNA modifications on 5' and 3' ends, allowing the recruitment of RNAse H and subsequent degradation of the messenger RNA. After transfection of tcDNA-ASO in patient-derived fibroblast cell lines, we show a strong decrease of HTT mRNA and protein levels. As a control, 2'O-methyl-RNA targeting the same region of HTT was also tested and did not induce a significant effect. tcDNA-ASO were also evaluated in vivo in the YAC128 mice, containing the full-length human HTT gene with 128 CAG repeat expansion. Single intracerebroventricular (ICV) injections of tcDNA induce a significant decrease of HTT messenger and protein levels in the cortex, hippocampus, striatum, and cerebellum of treated mice. tcDNA-ASO were found well distributed in the central nervous system (CNS) and show long lasting effect with protein levels still low, 12 weeks after a single ICV injection. This proof of concept study suggests the therapeutic potential of gapmer tcDNA ASO to downregulate huntingtin in vitro and in vivo.

Antisense-mediated exon skipping is one of the most promising therapeutic approaches for the treatment of Duchenne muscular dystrophy (DMD). In the past few years, this RNA-based strategy, mostly mediated by antisense oligonucleotides (AOs), has moved toward clinical evaluation, has demonstrated encouraging results, and has led the FDA to grant accelerated approval to one of these compounds recently. However significant clinical improvement in DMD patients has not been shown thus far, and AO-mediated exon skipping still faces major hurdles such as low efficacy in targeted tissues, poor cellular uptake, and relatively rapid clearance from circulation. These properties drive the need for repeated administrations in order to achieve a therapeutic response, with the negative consequence of accumulation in tissues and associated toxicity. To overcome these limitations, small nuclear RNAs (snRNAs) have been used to shuttle the antisense sequences, offering the advantage of a correct subcellular localization with pre-mRNAs and the potential for long-term correction when introduced into viral vectors such as adeno-associated virus (AAV) vectors. In this chapter, we review the development of the AAV-snRNA-mediated splicing modulation for DMD, focusing on the advantages offered by this technology over classical AOs as well as the challenges limiting their clinical application.

Antisense oligonucleotides (AONs) have been actively developed for more than 30 years as a form of molecular medicine and represent promising therapeutic tools for many disorders. Significant progress has been made toward their clinical development in particular for splice switching AONs for the treatment of neuromuscular disorders such as Duchenne muscular dystrophy (DMD). Many different chemistries of AONs can be used for splice switching modulation, and some of them have now reached regulatory approval. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake and sufficient therapeutic efficacy is difficult to achieve. Therefore, there is still a critical need to develop efficient AONs able to target all relevant tissues and international efforts are currently on going to advance new compounds or alternative chemistries with higher therapeutic potential. Here we describe the methods to evaluate the potency of tricyclo-DNA (tcDNA)-AONs, a novel class of AONs which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration (Goyenvalle et al., Nat Med 21:270-275, 2015; Goyenvalle et al., J Neuromuscul Dis 3:157-167, 2016; Relizani et al., Mol Ther Nucleic Acids 8:144-157, 2017; Robin et al., Mol Ther Nucleic Acids 7:81-89, 2017). We will focus on the preclinical evaluation of these tcDNA for DMD, specifically targeting the exon 51 of the human dystrophin gene. We will first detail methods to analyze their efficacy both in vitro in human myoblasts and in vivo in the hDMD and mdx52 mouse models and then describe means to evaluate their potential renal toxicity.

International audience; Antisense oligonucleotides (AONs) hold promise for therapeutic correction of many genetic diseases via exon skipping, and the first AON-based drugs have entered clinical trials for neuromuscular disorders. However, despite advances in AON chemistry and design, systemic use of AONs is limited because of poor tissue uptake, and recent clinical reports confirm that sufficient therapeutic efficacy has not yet been achieved. Here we present a new class of AONs made of tricyclo-DNA (tcDNA), which displays unique pharmacological properties and unprecedented uptake by many tissues after systemic administration. We demonstrate these properties in two mouse models of Duchenne muscular dystrophy (DMD), a neurogenetic disease typically caused by frame-shifting deletions or nonsense mutations in the gene encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, respiratory failure and neurocognitive impairment. Although current naked AONs do not enter the heart or cross the blood-brain barrier to any substantial extent, we show that systemic delivery of tcDNA-AONs promotes a high degree of rescue of dystrophin expression in skeletal muscles, the heart and, to a lesser extent, the brain. Our results demonstrate for the first time a physiological improvement of cardio-respiratory functions and a correction of behavioral features in DMD model mice. This makes tcDNA-AON chemistry particularly attractive as a potential future therapy for patients with DMD and other neuromuscular disorders or with other diseases that are eligible for exon-skipping approaches requiring whole-body treatment.

Antisense oligonucleotides (AONs) hold great promise for therapeutic splice-switching correction in many genetic diseases and in particular for Duchenne muscular dystrophy (DMD), where AONs can be used to reframe the dystrophin transcript and give rise to a partially deleted but yet functional dystrophin protein. Many different chemistries of AONs can be used for splice switching modulation, and some of them have been evaluated in clinical trials for DMD. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake, and sufficient therapeutic efficacy is difficult to achieve. Therefore, there is still a critical need to develop efficient AONs able to restore the expression of dystrophin in all relevant tissues and international efforts are currently on going to develop new compounds or alternative chemistries with higher therapeutic potential. Here, we describe the methods to evaluate the potency of antisense oligonucleotides, and in particular of tricyclo-DNA (tcDNA)-AONs, a novel class of AONs which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration. We focus on the most widely used mouse model for DMD, the mdx mouse and detail methods to analyze the skipping of the mouse exon 23 both in vitro in H2K mdx cells and in vivo in the mdx mouse model.

The use of splice-switching antisense therapy is highly promising, with a wealth of pre-clinical data and numerous clinical trials ongoing. Nevertheless, its potential to treat a variety of disorders has yet to be realized. The main obstacle impeding the clinical translation of this approach is the relatively poor delivery of antisense oligonucleotides to target tissues after systemic delivery. We are a group of researchers closely involved in the development of these therapies and would like to communicate our discussions concerning the validity of standard methodologies currently used in their pre-clinical development, the gaps in current knowledge and the pertinent challenges facing the field. We therefore make recommendations in order to focus future research efforts and facilitate a wider application of therapeutic antisense oligonucleotides., Fundación Ramón Areces and from Spanish Ministry of Economy and Competitiveness and European Regional Development Fund [grant reference SAF2013-43005-R]. B.S. was supported by a grant from the Tromsø Research Foundation. F.P.R. was supported by the Association Française contre les Myopathies (AFM-téléthon). M.A.D. was supported by Telethon Italia [grant reference GGP08244] and Italian Ministry of Health [grant reference GR-2008-1136933 and RF-2011-02347694]. S.L. was supported by The Dutch Duchenne Parent Project NL (DPP NL) and Association Française contre les Myopathies (AFM-téléthon). G.N.G. holds a Miguel Servet Fellowship from the ISCIII (grant reference CP14/00032] and a Fondo de Investigaciones Sanitarias Grant [grant reference PI15/01756], both are partfunded by the European Regional Development Fund (ERDF/FEDER). V.S. was supported by SKIP-NMD project, a European Community’s Seventh

In the context of future adeno-associated viral (AAV)-based clinical trials for Duchenne myopathy, AAV genome fate in dystrophic muscles is of importance considering the viral capsid immunogenicity that prohibits recurring treatments. We showed that AAV genomes encoding non-therapeutic U7 were lost from mdx dystrophic muscles within 3 weeks after intramuscular injection. In contrast, AAV genomes encoding U7ex23 restoring expression of a slightly shortened dystrophin were maintained endorsing that the arrest of the dystrophic process is crucial for maintaining viral genomes in transduced fibers. Indeed, muscles treated with low doses of AAV-U7ex23, resulting in sub-optimal exon skipping, displayed much lower titers of viral genomes, showing that sub-optimal dystrophin restoration does not prevent AAV genome loss. We also followed therapeutic viral genomes in severe dystrophic dKO mice over time after systemic treatment with scAAV9-U7ex23. Dystrophin restoration decreased significantly between 3 and 12 months in various skeletal muscles, which was correlated with important viral genome loss, except in the heart. Altogether, these data show that the success of future AAV-U7 therapy for Duchenne patients would require optimal doses of AAV-U7 to induce substantial levels of dystrophin to stabilize the treated fibers and maintain the long lasting effect of the treatment. © The American Society of Gene and Cell Therapy.

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.

RNA plays complex roles in normal health and disease and is becoming an important target for therapeutic intervention; accordingly, therapeutic strategies that modulate RNA function have gained great interest over the past decade. Antisense oligonucleotides (AOs) are perhaps the most promising strategy to modulate RNA expression through a variety of post binding events such as gene silencing through degradative or non-degradative mechanisms, or splicing modulation which has recently demonstrated promising results. However, AO technology still faces issues like poor cellular-uptake, low efficacy in target tissues and relatively rapid clearance from the circulation which means repeated injections are essential to complete therapeutic efficacy. To overcome these limitations, viral vectors encoding small nuclear RNAs have been engineered to shuttle antisense sequences into cells, allowing appropriate subcellular localization with pre-mRNAs and permanent correction. In this review, we outline the different strategies for antisense therapy mediated by viral vectors and provide examples of each approach. We also address the advantages and limitations of viral vector use, with an emphasis on their clinical application.

Summary Duchenne muscular dystrophy is a rare, progressive, muscle-wasting disease leading to severe disability and premature death. Treatment is currently symptomatic, but several experimental therapies are in development. Implemented care standards, validated outcome measures correlating with clinical benefit, and comprehensive information about the natural history of the disease are essential for regulatory approval of any treatment. However, for Duchenne muscular dystrophy and other rare diseases, these requirements are not always in place when potential therapies enter the clinical trial phase. A cooperative effort of stakeholders in Duchenne muscular dystrophy—including representatives from patients' groups, academia, industry, and regulatory agencies—is aimed at addressing this shortfall by identifying strategies to overcome challenges, developing the tools needed, and collecting relevant data. An open and constructive dialogue among European stakeholders has positively affected development of treatments for Duchenne muscular dystrophy; this approach could serve as a paradigm for development of treatments for rare diseases in general.

Antisense oligonucleotides are short nucleic acids designed to bind to specific messenger RNAs in order to modulate splicing patterns or inhibit protein translation. As such, they represent promising therapeutic tools for many disorders and have been actively developed for more than 20 years as a form of molecular medicine. Although significant progress has been made in developing these agents as drugs, they are yet not recognized as effective therapeutics and several hurdles remain to be overcome. Within the last few years, however, the prospect of successful oligonucleotides-based therapies has moved a step closer, in particular for Duchenne muscular dystrophy. Clinical trials have recently been conducted for this myopathy, where exon skipping is being used to achieve therapeutic outcomes. In this review, the recent developments and clinical trials using antisense oligonucleotides for Duchenne muscular dystrophy are discussed, with emphasis on the challenges ahead for this type of therapy, especially with regards to delivery and regulatory issues.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and recent clinical trials have demonstrated encouraging results. However, antisense oligonucleotide-mediated exon skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, poor cellular uptake and relatively rapid clearance from circulation, which means that repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, we previously proposed the use of small nuclear RNAs (snRNAs), especially U7snRNA to shuttle the antisense sequences after vectorization into adeno-associated virus (AAV) vectors. In this study, we report for the first time the efficiency of the AAV-mediated exon skipping approach in the utrophin/dystrophin double-knockout (dKO) mouse which is a very severe and progressive mouse model of DMD. Following a single intravenous injection of scAAV9-U7ex23 in dKO mice, near-normal levels of dystrophin expression were restored in all muscles examined, including the heart. This resulted in a considerable improvement of their muscle function and dystrophic pathology as well as a remarkable extension of the dKO mice lifespan. These findings suggest great potential for AAV-U7 in systemic treatment of the DMD phenotype.