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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

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.

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.

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.

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD but still faces personalized medicine challenges as different mutations found in DMD patients require skipping of different exons. However, 70% of DMD patients harbor dystrophin gene deletions in a mutation-rich area or "hot-spot" in the central genomic region. In this study, we have developed 11 different U7 small-nuclear RNA, to shuttle antisense sequences designed to mask key elements involved in the splicing of exons 45 to 55. We demonstrate that these constructs induce efficient exon skipping both in vitro in DMD patients' myoblasts and in vivo in human DMD (hDMD) mice and that they can be combined into a single vector to achieve a multi skipping of at least 3 exons. These very encouraging results provide proof of principle that efficient multiexon-skipping can be achieved using adeno-associated viral (AAV) vectors encoding multiple U7 small-nuclear RNAs (U7snRNAs), offering therefore very promising tools for clinical treatment of DMD. © The American Society of Gene and Cell Therapy.

Splice-modulation therapy aiming at correcting genetic defects by molecular manipulation of the premessenger RNA is a promising novel therapeutic approach for genetic diseases. In recent years, these new RNA based strategies, mostly mediated by antisense oligonucleotides (AO), have demonstrated encouraging results for muscular dystrophies, a heterogeneous group of genetic disorders characterized by muscle weakness and wasting. In particular, the clinical evaluation of antisense-mediated exon-skipping for the treatment of Duchenne muscular dystrophy has shown convincing data and therefore raised hopes and expectations for neuromuscular disorders therapy. However, AO-mediated splicing modulation still faces major hurdles such as low efficacy in specific tissues, poor cellular uptake and relatively rapid clearance from circulation, which means repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, small nuclear RNAs (snRNA) have been used to shuttle the antisense sequences, offering the advantage of a correct subcellular localization with pre-mRNAs and the potential of a permanent correction when introduced into viral vectors. Here we review the recent progress in the development of snRNA mediated splicing modulation for muscular dystrophies, focusing on the advantages offered by this technology over classical AOs but also the challenges limiting their clinical application.

Muscular dystrophies are a heterogeneous group of genetic disorders characterized by muscle weakness and wasting. Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, and although the molecular mechanisms of the disease have been extensively investigated since the discovery of the gene in 1986, there is currently no effective treatment. However, new gene-based therapies have recently emerged with particular noted advances in using conventional gene replacement strategies, RNA-based technology and pharmacological approaches. While the proof of principle has been demonstrated in animal models, several clinical trials have recently been undertaken to investigate the feasibility of these strategies in patients. In particular, antisense-mediated exon skipping has shown encouraging results and holds promise for the treatment of dystrophic muscle. Here, we summarize the recent progress in therapeutic approaches to muscular dystrophies, with an emphasis on gene therapy and exon skipping for DMD.

Numerous strategies are under development for the correction of deleterious effects of mutations in muscular dystrophies, and these strategies must be validated in compelling models. Cellular models seem straightforward to set up; however, the proliferative capacity of muscle cells isolated from dystrophic patients is limited, and in addition it is difficult to envisage the use of large muscle biopsies from patients to obtain enough cells for ex vivo assessments. To overcome these problems, we have devised a strategy to obtain, from a patient with Duchenne muscular dystrophy (DMD), an inexhaustible source of myogenic progenitor cells with a deletion of exons 49 and 50 in the dystrophin gene. Starting material consisted of dermal fibroblasts isolated from a skin biopsy taken in a noninvasive way. These fibroblasts were first immortalized by telomerase gene transfer. Subsequent cell lines were converted into myogenic cells by means of a lentiviral vector encoding an inducible MyoD construct. Before myogenic induction, engineered DMD fibroblasts were able to proliferate infinitely. Under induction conditions, they were converted into myogenic cells, which differentiated into large multinucleated myotubes. We used these DMD fibroblast cell lines to assess dystrophin rescue by using engineered U7 small nuclear RNAs harboring antisense sequences required to restore an in-frame dystrophin mRNA by skipping exon 51. Further molecular analyses showed dystrophin rescue ex vivo as well as in vivo after engrafting of treated cells into regenerating muscles in immunodeficient mice.

Exon-skipping AAV1-U7-associated therapy is a promising treatment for Duchenne muscular dystrophy (DMD). We have shown earlier that the newly rescued dystrophin protein is stably expressed for months in mice and dogs, and does not induce immune rejection of transduced fibers. In this study, we used the dystrophic mdx mouse as a preclinical model to characterize the immune response to the adeno-associated virus 1 (AAV1) vector, and tested the feasibility of administering multiple AAV1 injections to extend the treatment to several muscles. We found that re-injections of AAV1 vector are compromised as early as 3 days after the first injection, coincident with a rapid increase in AAV1-specific immunoglobulin M (IgM) and IgG in the serum. Adoptive transfer of immune sera confirmed the rapid appearance of an AAV1 neutralization activity, and experiments with immunoglobulin-deficient (microKO) mice proved that antibodies (Abs) are the only effectors responsible for AAV1-U7 elimination. It is important to note, however, that the AAV2 vector still generated an adverse immune response in microKO mice. By blocking the T-B crosstalk with anti-CD40 Abs and CTLA4/Fc fusion protein, we found that a mere 5 days of immunomodulation treatment was sufficient to totally abrogate the formation of anti-AAV1 Abs and to allow for the correction of muscular dystrophy in multiple muscles, provided the treatment was administered during each challenge.

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> Les dystrophinopathies sont des affections liees a des anomalies de la dystrophine, proteine sub-sarcolemmique de 427 kDa. Celle-ci est soit absente (dystrophie musculaire progressive et severe de Duchenne), soit presente mais diminuee et/ou alteree qualitativement (dystrophies moins severes de type Becker). Cette heterogeneite clinique est sous-tendue par une heterogeneite des mutations dans le gene DMD (2,4 Mb, 79 exons) qui est l’objet de mutations tres variables que l’on peut regrouper en deux categories affectant differemment la synthese de la dystrophine : (1) les grands remaniements touchant un ou plusieurs exons (deletions dans 60%70% des cas, duplications dans 10%-15 % des cas); (2) les «petites» mutations intraou juxta-exoniques (miniinsertions ou deletions, mutations ponctuelles de type non-sens direct, mutations d’epissage). En regle generale, la forme de Duchenne est due a des mutations nulles, incompatibles avec une quelconque production de dystrophine (mutations interrompant le cadre de lecture, comme les mutations non-sens direct, les deletions ou insertions creant un codon non-sens premature). Les formes moins severes de type Becker sont dues a des mutations compatibles avec la production d’une certaine quantite de dystrophine. En ce qui concerne les grandes deletions internes, de loin les plus frequentes, la severite du phenotype (Duchenne ou Becker) n’est pas conditionnee par leur etendue mais par l’impact sur le cadre de lecture. De tres grandes deletions peuvent n’entrainer qu’un tableau clinique peu severe si le cadre de lecture est preserve. En effet, la distribution des 11 055 nucleotides de la sequence codante de l’isoforme musculaire de la dystrophine (Dp427 = 427kDa) sur les 79 exons du gene DMD ne coincide pas regulierement avec le decoupage en triplets. Ainsi, la dystrophine, avec sa structure modulaire, et notamment son domaine central constitue de 24 repetitions de motifs de type spectrine (109 acides amines), est une proteine tolerant l’ablation de certaines regions internes, pourvu que la phase de la sequence codante finale soit preservee. Cela a conduit au concept de modeles reduits mais encore fonctionnels de la dystrophine: mini-dystrophine, amputee de 46 % de la sequence peptidique [1] et micro-dystrophine, amputee de 60 % [2], tres utilisees dans les essais de therapie genique par transfert de gene. Compte tenu de l’organisation des exons du gene DMD, on concoit qu’une phase abolie par une mutation (deletion ou mutation ponctuelle) puisse etre retablie par l’exclusion d’un ou plusieurs exons si la phase locale s’y prete. C’est ce qu’on appelle le saut d’exon therapeutique (SET) (en anglais, exon skipping). D’ou l’idee d’intervenir sur le processus d’epissage pour empecher l’incorporation de l’exon ou des exons choisis. Ces sauts d’exon peuvent etre induits par des oligonucleotides antisens (modifies pour resister aux RNases), complementaires de sequences-cles qui president a l’epissage du transcrit primaire. Des 1991, M. Matsuo [3] avait insiste sur l’importance des phenomenes d’epissage dans le determinisme et la therapeutique des dystrophinopathies. Plus recemment, d’autres groupes se sont engages dans la voie de l’epissotherapie, comme alternative a la therapie genique classique qui n’a pas encore remporte les succes escomptes [4-6]. Une restauration de la synthese de dystrophine musculaire chez la souris mdx par exclusion post-transcriptionnelle de l’exon porteur de la mutation (stop sur l’exon 23) a ete rapportee par plusieurs DERNIERE HEURE

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused, in most cases, by the complete absence of the 427 kDa cytoskeletal protein, dystrophin. There is no effective treatment, and affected individuals die from respiratory failure and cardiomyopathy by age 30. Here, we investigated whether cardiomyopathy could be prevented in animal models of DMD by increasing diaphragm utrophin or dystrophin expression and thereby restoring diaphragm function. In a transgenic mdx mouse, where utrophin was over expressed in the skeletal muscle and the diaphragm, but not in the heart, we found cardiac function, specifically right and left ventricular ejection fraction as measured using in vivo magnetic resonance imaging, was restored to wild-type levels. In mdx mice treated with a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) that resulted in high levels of dystrophin restoration in the skeletal muscle and the diaphragm only, cardiac function was also restored to wild-type levels. In dystrophin/utrophin-deficient double-knockout (dKO) mice, a more severely affected animal model of DMD, treatment with a PPMO again produced high levels of dystrophin only in the skeletal muscle and the diaphragm, and once more restored cardiac function to wild-type levels. In the dKO mouse, there was no difference in heart function between treatment of the diaphragm plus the heart and treatment of the diaphragm alone. Restoration of diaphragm and other respiratory muscle function, irrespective of the method used, was sufficient to prevent cardiomyopathy in dystrophic mice. This novel mechanism of treating respiratory muscles to prevent cardiomyopathy in dystrophic mice warrants further investigation for its implications on the need to directly treat the heart in DMD.

International audience; Dystrophin, the cytoskeletal protein whose defect is responsible for Duchenne muscular dystrophy (DMD), is normally expressed in both muscles and brain. Genetic loss of brain dystrophin in the mdx mouse model of DMD reduces the capacity for type A gamma-aminobutyric acid (GABA(A))-receptor clustering in central inhibitory synapses, which is thought to be a main molecular defect leading to brain and cognitive alterations in this syndrome. U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors have proven efficient after intramuscular injection to induce skipping of the mutated exon 23 and rescue expression of a functional dystrophin-like product in muscle tissues of mdx mice in vivo. Here, we report that intrahippocampal injection of a single dose of rAAV2/1-U7 can rescue substantial levels of brain dystrophin expression (15-25%) in mdx mice for months. This is sufficient to completely restore GABA(A)-receptor clustering in pyramidal and dendritic layers of CA1 hippocampus, suggesting exon-skipping strategies offer the prospect to investigate and correct both brain and muscle alterations in DMD. This provides new evidence that in the adult brain dystrophin is critical for the control of GABA(A)-receptor clustering, which may have an important role in activity-dependent synaptic plasticity in hippocampal circuits.

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 because of its capacity to correct the reading frame and restore dystrophin expression, which has been demonstrated in vitro and in vivo. In particular, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have recently been shown to induce widespread high levels of dystrophin expression in the mdx mouse model. Here, we report the efficiency of the PPMO-mediated exon-skipping approach in the utrophin/dystrophin double-knockout mouse (dKO) mouse, which is a much more severe and progressive mouse model of DMD. Repeated intraperitoneal (i.p.) injections of a PPMO targeted to exon 23 of dystrophin pre-mRNA in dKO mice induce a near-normal level of dystrophin expression in all muscles examined, except for the cardiac muscle, resulting in a considerable improvement of their muscle function and dystrophic pathology. These findings suggest great potential for PPMOs in systemic treatment of the DMD phenotype.

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. In most cases, the open-reading frame is disrupted which results in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and has recently been shown to correct the reading frame and restore dystrophin expression in vitro and in vivo. Specific exon skipping can be achieved using synthetic oligonucleotides or viral vectors encoding modified small nuclear RNAs (snRNAs), by masking important splicing sites. In this study, we demonstrate that enhanced exon skipping can be induced by a U7 snRNA carrying binding sites for the heterogeneous ribonucleoprotein A1 (hnRNPA1). In DMD patient cells, bifunctional U7 snRNAs harboring silencer motifs induce complete skipping of exon 51, and thus restore dystrophin expression to near wild-type levels. Furthermore, we show the efficacy of these constructs in vivo in transgenic mice carrying the entire human DMD locus after intramuscular injection of adeno-associated virus (AAV) vectors encoding the bifunctional U7 snRNA. These new constructs are very promising for the optimization of therapeutic exon skipping for DMD, but also offer powerful and versatile tools to modulate pre-mRNA splicing in a wide range of applications.

SummaryDuchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intramuscular and intra-arterial delivery of genetically corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recovery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.

Objectives: The objective of this study was to assess the utility of the gene expression profiling technique for the preclinical evaluation of drug efficacy and safety, taking a new therapeutic approach for Duchenne muscular dystrophy (DMD) as an example. Methods: Muscles from dystrophin-deficient (mdx) mice, a well-characterized animal model for DMD, were injected with antisense constructs that restore the open reading frame in the Dmd gene. Synthetic antisense oligonucleotides (AONs) complexed with different carriers to enhance cellular uptake and recombinant adeno-associated virus (rAAV)-expressed antisense sequences were evaluated. Muscular gene expression profiles were analyzed on oligonucleotide microarrays. Results: Polyethylenimine (PEI)-complexed AONs restored the reading frame slightly more effectively than uncomplexed, F127- or Optison™-complexed AONs. However, PEI induced the expression of many immune genes, reflecting an aggravation of the inflammation present in untreated mdx mice. Expression profiles in Optison and F127-injected muscles were similar to those of saline treated muscles, implying that these carriers did not evoke adverse responses. Due to moderate levels of exon skipping, a significant shift toward wild-type expression levels was not detected. Injection with rAAV vectors resulted in much higher production of dystrophin and greatly improved the histological appearance of the muscle. Depending on the efficacy of the treatment, the expression of genes previously shown to be elevated in muscular dystrophies, partly or completely returned to wild-type expression levels. Reductions in inflammation and fibrosis were among the most prominent changes observed. Conclusion: Expression profiling is a powerful tool for the evaluation of both desired and adverse effects of new pharmacological therapies. It is sensitive and detects changes that are not histologically visible. In addition, its ability to simultaneously monitor a large number of different biological processes not only reduces the number of different assays required in preclinical research and clinical trials, but may also assist in the early detection of potential side effects.

International audience; Muscular dystrophies are a genetically and phenotypically heterogeneous group of degenerative muscle diseases. A subset of them are due to genetic deficiencies in proteins which form the dystrophin-associated complex at the membrane of the myofibers. In this report, we utilized recombinant adeno-associated virus containing a U7 cassette carrying an antisense sequence aimed at inducing exon skipping of the dystrophin gene or containing the alpha-sarcoglycan gene to alleviate the dystrophic phenotype of the mdx and Sgca- null mice, respectively. As these diseases are characterized by cycle of degeneration/regeneration, we postulated that a reporter gene coadministered at the time of the treatment would make it possible to follow the extent of muscle repair. We observed that the murine secreted alkaline phosphatase (muSeAP) level was very much lower in these animal models than in normal mice. Upon treatment of the dystrophic muscle by gene transfer, the level of muSeAP was restored and correlated with the expression of the therapeutic transgene and with the level of muscle improvement. The system described here provides a simple and noninvasive procedure for monitoring the outcome of a therapeutic strategy involving cell survival.

Top of pageAbstract Most cases of Duchenne muscular dystrophy (DMD) are caused by dystrophin gene mutations that disrupt the mRNA reading frame. In some cases, forced exclusion of a single exon can restore the reading frame, given rise to a shorter, but still functional dystrophin protein (so called quasi-dystrophin). One potential treatment of the disorder has utilized antisense oligoribonucleotide (AO) to induce removal of disease-associated exons during pre-mRNA processing. Indeed, this approach has been successfully used in DMD cells in vitro with antisense sequences against splice junctions of exon 51. Skipping of this exon would theoretically restore a functional quasi- dystrophin in a significant subset of DMD patients with |[Delta]|45-50,|[Delta]|47-50,|[Delta]|48-50, |[Delta]|49-50, |[Delta]|50 and |[Delta]|52 genotypes. However, since the AO are not self-renewed, they can not achieve long term correction. To overcome this limitation, we have introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized them in AAV and lentiviral vectors. We have designed AAV and lentiviral vectors harboring chimeric U7 snRNA carrying antisense sequences against exon 51 of the human dystrophin gene (U7-ex51). Lentiviral vectors expressing this U7-ex51 were tested on human myoblasts, whereas AAV vectors were injected in the transgenic hDMD mice (carrying the human dystrophin gene). We confirmed the skipping of the exon 51 in vitro in human myoblasts after transduction with the lentiviral vector encoding U7-ex51 by RT-PCR. We also detected the skipping of the exon 51 after intramuscular injection of an AAV-U7ex51 vector in the transgenic hDMD mouse. We have also tested the efficacy of these vectors to restore dystrophin expression in myoblasts from patients with |[Delta]|49-50 and |[Delta]|52 deletions. In this study, we provide evidence that efficient skipping of exon 51 can be achieved in human cells and also in vivo after intramuscular injection in a transgenic hDMD mice through U7snRNA shuttle. These results offer very promising tools for clinical treatment of DMD.

Most mutations in the dystrophin gene create a frameshift or a stop in the mRNA and are associated with severe Duchenne muscular dystrophy. Exon skipping that naturally occurs at low frequency sometimes eliminates the mutation and leads to the production of a rescued protein. We have achieved persistent exon skipping that removes the mutated exon on the dystrophin messenger mRNA of the mdx mouse, by a single administration of an AAV vector expressing antisense sequences linked to a modified U7 small nuclear RNA. We report the sustained production of functional dystrophin at physiological levels in entire groups of muscles and the correction of the muscular dystrophy.

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 because of its capacity to correct the reading frame and restore dystrophin expression in vitro and in vivo. This approach has now been tested in clinical trials with two different chemistries, the 2′O-Methyl phosphorothioate oligoribonucelotides (2′OMe) and the phosphorodiamidate morpholino oligomers (PMO) and has demonstrated encouraging results. However, AO-mediated exon-skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, no crossing of the blood brain barrier, poor cellular uptake and relative rapid clearance from circulation. To overcome these limitations, we investigated the therapeutic potential of a new class of conformationally constrained DNA analogues: the tricyclo-DNAs (Tc-DNA). In this study, we demonstrate widespread restoration of dystrophin in the mdx mouse following systemic injections of Tc-DNA targeting the donor splice site of the dystrophin exon 23. Remarkably, this treatment also leads to restoration of dystrophin in the cardiac muscle and detection of exon skipping in the brain. Moreover, we also show the therapeutic benefit of Tc-DNA treatment in the severely affected utrophin/dystrophin double-knockout mouse (dKO) which is a much more severe and progressive mouse model of DMD. Tc-DNA injections result in significant improvement of their muscle function and dystrophic pathology, by preventing rachiokyphosis and contractures and improving their motility, as assessed by activity monitoring. These findings indicate that Tc-DNA exhibit therapeutic, drug-like effects not only in relatively benign mdx mouse model but also in very severe DMD phenotype of dKO mouse, suggesting great potential for these compounds in the systemic treatment of DMD.

Our goal is to evaluate the consequences of therapeutic gene transfer in the brains of adult mdx mice with memory deficits. We will try to compensate the deficits of mdx mice, by means of intracerebral administrations of vectorized antisense sequences that are potentially able to eliminate the mutated exon 23 of the dystrophin gene in this mutant, thereby allowing a local restoration of brain-dystrophin expression. We have recently developed and validated this technology and obtained a complete, functional and enduring restoration of dystrophin in the skeletal muscle of mdx mice [Goyenvalle et al., 2004]. AAV vector considerably enhanced the proper subcellular localization of antisense sequences and their inclusion into the spliceosome. Transfection of snRNA into target cells is facilitated by the use of gene-tranfer vectors derived from parvovirus (AAV) or lentivirus (HIV). These types of vectors have been shown to be particularly efficient for gene transfer into pyramidal neurons of the hippocampus.We show here that rescue of almost the entire dorsal hippocampus has been achieved by a bilateral intracerebral stereotaxic administration of an AAV-vector expressing antisense sequences linked to a modified U7 small nuclear RNA (snRNA). Restoration of dystrophin expression can be obtained in CA1 pyramidal cells of adult mdx mice after stereotaxic administration of the AAV-vector expressing the appropriate antisense sequences. This result already suggests that the reversal of genetic alterations can be achieved in the mature brain. To confirm and quantify the restoration of dystrophin expression, mRNA analyses and protein detection immunohistochemistry were carried out on serial brain tissue sections. The DNA sequence confirmed that the 688 bp band resulting from RT-PCR corresponds to the exon 23-skipped mRNA.We will now determine whether this treatment may compensate the long-term memory deficits displayed by mdx mice in object recognition tests and massed-training in the water maze. We will also establish the impact of the treatment on the neural mechanisms thought to underly the cognitive deficits associated with dystrophin loss (e.g., LTP, GABAA-receptor clustering, expression of dystrophin-associated proteins). Finally we will determine whether the efficacy of this invasive therapeutic approach may be potentiated by experimental stimulation of functional brain plasticity or by pharmacological modulation of GABAergic transmission.

Top of pageAbstract In Duchenne Muscular Dystrophy (DMD), the lack of Dystrophin results in a series of catastrophic events that lead to muscle fiber death and muscle wasting. Dystrophin is a modular protein with a central rod domain composed of 24 spectrin-like repeats and truncated proteins missing some of these repeats can be fully functional or at least partly deficient as seen in patients with mild (Becker) forms of DMD. In a majority of severe DMD patients that presents out of frame alterations of the dystrophin transcript, a translational open reading frame would be restored by eliminating selected exon(s) from the pre mRNA. Exon skipping naturally occurs during dystrophin mRNA processing giving rise to rare revertant fibers that contain shortened proteins. It can be forced using antisense sequences delivered as synthetic oligonucleotides, or attached to a small nuclear (sn) RNA, notably U7. A |[ldquo]|double-target|[rdquo]| U7SmOPT containing antisense sequences from introns 22 and 23 (U7-SD23/BP22 ) was introduced in an AAV vector and packaged as an AAV2/1 pseudotype. Adult mdx mice were injected into the Tibialis Anterior, or submitted to whole limb arterial perfusion, and analysed between 2 and 13 weeks. Dystrophin transcripts missing exon 23 were detected by RT-PCR, representing 15% of the amplified material at two weeks and becoming the major species (60 to 70%) at one month. The Dystrophin protein was readily detected both by Western blot on muscle extracts and by immunofluorescence on tissue sections were restoration of the Dystrophin Associated Complex was also documented. The levels of rescued dystrophin was 3 % of normal at 2 weeks and 50 to 80% thereafter. The treated muscles were shown to recover normal contractile and mechanical properties by measuring resistance to eccentric contractions. Fiber resistance to exercise-induced damage was also restored, as seen using Evans Blue exclusion. The level and stability of U7-induced exon skipping that we observe in vivo in the context of an AAV vector is unparalleled, and provides a strong rational for the development of this approach in the clinic.

International audience; Duchenne muscular dystrophy (DMD) is a neurodevelopmental disorder primarily caused by the loss of the full-length Dp427 dystrophin in both muscle and brain. The basis of the central comorbidities in DMD is unclear. Brain dystrophin plays a role in the clustering of central gamma-aminobutyric acid A receptors (GABAARs), and its loss in the mdx mouse alters the clustering of some synaptic subunits in central inhibitory synapses. However, the diversity of GABAergic alterations in this model is still fragmentary. In this study, the analysis of in vivo PET imaging of a benzodiazepine-binding site radioligand revealed that the global density of central GABAARs is unaffected in mdx compared with WT mice. In contrast, semi-quantitative immunoblots and immunofluorescence confocal imaging in tissue sections revealed complex and differential patterns of alterations of the expression levels and/or clustered distribution of a variety of synaptic and extrasynaptic GABAAR subunits in the hippocampus, cerebellum, cortex, and spinal cord. Hence, dystrophin loss not only affects the stabilization of synaptic GABAARs but also influences the subunit composition of GABAARs subtypes at both synaptic and extrasynaptic sites. This study provides new molecular outcome measures and new routes to evaluate the impact of treatments aimed at compensating alterations of the nervous system in DMD.